Greg Barritt

A small component of the endoplasmic reticulum is required for store-operated Ca2+ channel activation in liver cells: evidence from studies using TRPV1 and taurodeoxycholic acid

Publisher: Portland Press Ltd.

Date: 25-02-2009

DOI: 10.1042/BJ20081052

Abstract: The question of whether the activation of SOCs (store-operated Ca2+ channels) requires the whole or part of the ER (endoplasmic reticulum) has not been fully resolved. The role of a putative sub-compartment of the ER in SOC activation in liver cells was investigated using ectopically expressed TRPV1 (transient receptor potential vanilloid 1), a non-selective cation channel, and TDCA (taurodeoxycholic acid), an activator of SOCs, to release Ca2+ from different regions of the ER. TRPV1 was expressed in the ER and in the plasma membrane. The amount of Ca2+ released from the ER by a TRPV1 agonist, measured using fura-2, was the same as that released by a SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) inhibitor, indicating that TRPV1 agonist-sensitive stores substantially overlap with SERCA inhibitor-sensitive stores. In contrast with SERCA inhibitors, TRPV1 agonists did not activate store-operated Ca2+ entry. These findings were confirmed by patch-cl recording. Using FFP-18, it was shown that SERCA inhibitors release Ca2+ from the ER located closer to the plasma membrane than the region from which TRPV1 agonists release Ca2+. In contrast with SERCA inhibitors, TRPV1 agonists did not induce a redistribution of STIM1 (stromal interaction molecule 1). TDCA caused the release of Ca2+ from the ER, which was detected by FFP-18 but not by fura-2, and a redistribution of STIM1 to puncta similar to that caused by SERCA inhibitors. It is concluded that in liver cells, Ca2+ release from a small component of the ER located near the plasma membrane is required to induce STIM1 redistribution and SOC activation.

Phospholipase C-γ1 is required for the activation of store-operated Ca2+ channels in liver cells

Publisher: Portland Press Ltd.

Date: 27-06-2007

DOI: 10.1042/BJ20061762

Abstract: Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-γ (phospholipase C-γ) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-γ-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-γ has not been defined clearly. The aim of the present study was to determine if PLC-γ is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-γ1 caused a reduction (by approx. 70%) in the PLC-γ1 protein expression, with maximal effect at 72–96 h. This was associated with a decrease (by approx. 60%) in the litude of the ISOC (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP3 or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the ISOC litude. Immunoprecipitation of PLC-γ1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-γ1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.

TRPM2 channels mediate acetaminophen-induced liver damage

Publisher: Proceedings of the National Academy of Sciences

Date: 03-02-2014

DOI: 10.1073/PNAS.1322657111

Abstract: Acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. There are limited options for early treatment. Acetaminophen liver toxicity leads to the formation of reactive oxygen and nitrogen species which cause an increase in intracellular Ca 2+ and hepatocellular death. We show that acetaminophen-induced liver toxicity depends on Transient Receptor Potential Melanostatine 2 (TRPM2) cation channels in hepatocytes, which are activated in response to oxidative stress and are responsible for Ca 2+ overload. Lack of TRPM2 channels in hepatocytes or their pharmacological inhibition protects liver from acetaminophen toxicity. This provides evidence that TRPM2 may present a potential therapeutic target for treatment of oxidative-stress related liver diseases.

Regulation of Orai1/STIM1 mediated ICRAC by intracellular pH

Publisher: Springer Science and Business Media LLC

Date: 29-08-2017

DOI: 10.1038/S41598-017-06371-0

Abstract: Ca 2+ release activated Ca 2+ (CRAC) channels composed of two cellular proteins, Ca 2+ -sensing stromal interaction molecule 1 (STIM1) and pore-forming Orai1, are the main mediators of the Ca 2+ entry pathway activated in response to depletion of intracellular Ca 2+ stores. Previously it has been shown that the litude of CRAC current (I CRAC ) strongly depends on extracellular and intracellular pH. Here we investigate the intracellular pH (pH i ) dependence of I CRAC mediated by Orai1 and STIM1ectopically expressed in HEK293 cells. The results indicate that pH i affects not only the litude of the current, but also Ca 2+ dependent gating of CRAC channels. Intracellular acidification changes the kinetics of I CRAC , introducing prominent re-activation component in the currents recorded in response to voltage steps to strongly negative potentials. I CRAC with similar kinetics can be observed at normal pH i if the expression levels of Orai1 are increased, relative to the expression levels of STIM1. Mutations in the STIM1 inactivation domain significantly diminish the dependence of I CRAC kinetics on pH i , but have no effect on pH i dependence of I CRAC litude, implying that more than one mechanism is involved in CRAC channel regulation by intracellular pH.

TRPM2 Non-Selective Cation Channels in Liver Injury Mediated by Reactive Oxygen Species

Publisher: MDPI AG

Date: 03-08-2021

DOI: 10.3390/ANTIOX10081243

Abstract: TRPM2 channels admit Ca2+ and Na+ across the plasma membrane and release Ca2+ and Zn2+ from lysosomes. Channel activation is initiated by reactive oxygen species (ROS), leading to a subsequent increase in ADP-ribose and the binding of ADP-ribose to an allosteric site in the cytosolic NUDT9 homology domain. In many animal cell types, Ca2+ entry via TRPM2 channels mediates ROS-initiated cell injury and death. The aim of this review is to summarise the current knowledge of the roles of TRPM2 and Ca2+ in the initiation and progression of chronic liver diseases and acute liver injury. Studies to date provide evidence that TRPM2-mediated Ca2+ entry contributes to drug-induced liver toxicity, ischemia–reperfusion injury, and the progression of non-alcoholic fatty liver disease to cirrhosis, fibrosis, and hepatocellular carcinoma. Of particular current interest are the steps involved in the activation of TRPM2 in hepatocytes following an increase in ROS, the downstream pathways activated by the resultant increase in intracellular Ca2+, and the chronology of these events. An apparent contradiction exists between these roles of TRPM2 and the role identified for ROS-activated TRPM2 in heart muscle and in some other cell types in promoting Ca2+-activated mitochondrial ATP synthesis and cell survival. Inhibition of TRPM2 by curcumin and other “natural” compounds offers an attractive strategy for inhibiting ROS-induced liver cell injury. In conclusion, while it has been established that ROS-initiated activation of TRPM2 contributes to both acute and chronic liver injury, considerable further research is needed to elucidate the mechanisms involved, and the conditions under which pharmacological inhibition of TRPM2 can be an effective clinical strategy to reduce ROS-initiated liver injury.

Store-operated Ca2+ channels and Stromal Interaction Molecule 1 (STIM1) are targets for the actions of bile acids on liver cells

Publisher: Elsevier BV

Date: 05-2008

DOI: 10.1016/J.BBAMCR.2008.02.011

Abstract: Cholestasis is a significant contributor to liver pathology and can lead to primary sclerosis and liver failure. Cholestatic bile acids induce apoptosis and necrosis in hepatocytes but these effects can be partially alleviated by the pharmacological application of choleretic bile acids. These actions of bile acids on hepatocytes require changes in the release of Ca(2+) from intracellular stores and in Ca(2+) entry. However, the nature of the Ca(2+) entry pathway affected is not known. We show here using whole cell patch cl experiments with H4-IIE liver cells that taurodeoxycholic acid (TDCA) and other choleretic bile acids reversibly activate an inwardly-rectifying current with characteristics similar to those of store-operated Ca(2+) channels (SOCs), while lithocholic acid (LCA) and other cholestatic bile acids inhibit SOCs. The activation of Ca(2+) entry was observed upon direct addition of the bile acid to the incubation medium, whereas the inhibition of SOCs required a 12 h pre-incubation. In cells loaded with fura-2, choleretic bile acids activated a Gd(3+)-inhibitable Ca(2+) entry, while cholestatic bile acids inhibited the release of Ca(2+) from intracellular stores and Ca(2+) entry induced by 2,5-di-(tert-butyl)-1,4-benzohydro-quinone (DBHQ). TDCA and LCA each caused a reversible redistribution of stromal interaction molecule 1 (STIM1, the endoplasmic reticulum Ca(2+) sensor required for the activation of Ca(2+) release-activated Ca(2+) channels and some other SOCs) to puncta, similar to that induced by thapsigargin. Knockdown of Stim1 using siRNA caused substantial inhibition of Ca(2+)-entry activated by choleretic bile acids. It is concluded that choleretic and cholestatic bile acids activate and inhibit, respectively, the previously well-characterised Ca(2+)-selective hepatocyte SOCs through mechanisms which involve the bile acid-induced redistribution of STIM1.

The predominant role of IP3 type 1 receptors in activation of store-operated Ca2+ entry in liver cells

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.BBAMEM.2010.12.013

Abstract: Physiologically, hormone induced release of Ca²+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP₃) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP₃ receptors act as channels, releasing Ca²+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca²+ stores leads to activation of store-operated Ca²+ channels on the plasma membrane which replenishes lost Ca²+ and sustain Ca²+ signalling. There are three isoforms of IP₃ receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca²+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca²+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca²+ channels, and Ca²+ release activated Ca²+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP₃ receptor but not the others. Here we show that H4IIE liver cells express all three types of IP₃ receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca²+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca²+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP₃ receptors.

Steatosis inhibits liver cell store-operated Ca2+ entry and reduces ER Ca2+ through a protein kinase C-dependent mechanism

Publisher: Portland Press Ltd.

Date: 20-02-2015

DOI: 10.1042/BJ20140881

Abstract: Lipid accumulation in hepatocytes can lead to non-alcoholic fatty liver disease (NAFLD), which can progress to non-alcoholic steatohepatitis (NASH) and Type 2 diabetes (T2D). Hormone-initiated release of Ca2+ from the endoplasmic reticulum (ER) stores and subsequent replenishment of these stores by Ca2+ entry through SOCs (store-operated Ca2+ channels SOCE) plays a critical role in the regulation of liver metabolism. ER Ca2+ homoeostasis is known to be altered in steatotic hepatocytes. Whether store-operated Ca2+ entry is altered in steatotic hepatocytes and the mechanisms involved were investigated. Lipid accumulation in vitro was induced in cultured liver cells by amiodarone or palmitate and in vivo in hepatocytes isolated from obese Zucker rats. Rates of Ca2+ entry and release were substantially reduced in lipid-loaded cells. Inhibition of Ca2+ entry was associated with reduced hormone-initiated intracellular Ca2+ signalling and enhanced lipid accumulation. Impaired Ca2+ entry was not associated with altered expression of stromal interaction molecule 1 (STIM1) or Orai1. Inhibition of protein kinase C (PKC) reversed the impairment of Ca2+ entry in lipid-loaded cells. It is concluded that steatosis leads to a substantial inhibition of SOCE through a PKC-dependent mechanism. This enhances lipid accumulation by positive feedback and may contribute to the development of NASH and insulin resistance.

Increased expression of peroxiredoxin 1 and identification of a novel lipid‐metabolizing enzyme in the early phase of liver ischemia reperfusion injury

Publisher: Wiley

Date: 17-10-2011

DOI: 10.1002/PMIC.201100053

Abstract: Warm ischemia reperfusion (IR) injury of the liver is associated with changes in the expression and/or post-translational modification of numerous proteins. Only a few of these have been identified. We used 2-D DIGE to identify cytosolic proteins altered in the early stage of IR in an established rat model of segmental hepatic ischemia. Proteins in 18 abundant spots altered by IR were identified by LC-MS/MS and Western blot. Many identified proteins were enzymes involved in glucose and lipid metabolism. Isoamyl acetate-hydrolysing esterase 1 homolog, not previously characterized in liver, was also identified. A threefold increase in peroxiredoxin 1 (Prx1) and its oxidized forms was observed as was an increase in Prx1 mRNA. Peroxiredoxins and their overoxidation have previously been associated with IR. In contrast to other studies, we did not detect typical overoxidation of Prx1 on the peroxidatic cysteine (Cys(52)). Instead, we identified novel overoxidation of the resolving cysteine (Cys(173)) residue by LC-MS/MS. Our results show that a rapid increase in Prx1 expression is associated with the early phase of IR of the liver, likely contributing to mechanisms that protect the liver against IR damage. Additionally, we have revealed a potential role in liver for a novel lipid-metabolizing enzyme.

Targeting Ca2+ Signaling in the Initiation, Promotion and Progression of Hepatocellular Carcinoma

Publisher: MDPI AG

Date: 24-09-2020

DOI: 10.3390/CANCERS12102755

Abstract: Hepatocellular carcinoma (HCC) is a considerable health burden worldwide and a major contributor to cancer-related deaths. HCC is often not noticed until at an advanced stage where treatment options are limited and current systemic drugs can usually only prolong survival for a short time. Understanding the biology and pathology of HCC is a challenge, due to the cellular and anatomic complexities of the liver. While not yet fully understood, liver cancer stem cells play a central role in the initiation and progression of HCC and in resistance to drugs. There are approximately twenty Ca2+-signaling proteins identified as potential targets for therapeutic treatment at different stages of HCC. These potential targets include inhibition of the self-renewal properties of liver cancer stem cells HCC initiation and promotion by hepatitis B and C and non-alcoholic fatty liver disease (principally involving reduction of reactive oxygen species) and cell proliferation, tumor growth, migration and metastasis. A few of these Ca2+-signaling pathways have been identified as targets for natural products previously known to reduce HCC. Promising Ca2+-signaling targets include voltage-operated Ca2+ channel proteins (liver cancer stem cells), inositol trisphosphate receptors, store-operated Ca2+ entry, TRP channels, sarco/endoplasmic reticulum (Ca2++Mg2+) ATP-ase and Ca2+/calmodulin-dependent protein kinases. However, none of these Ca2+-signaling targets has been seriously studied any further than laboratory research experiments. The future application of more systematic studies, including genomics, gene expression (RNA-seq), and improved knowledge of the fundamental biology and pathology of HCC will likely reveal new Ca2+-signaling protein targets and consolidate priorities for those already identified.

Properties of Orai1 mediated store‐operated current depend on the expression levels of STIM1 and Orai1 proteins

Publisher: Wiley

Date: 12-06-2009

DOI: 10.1113/JPHYSIOL.2009.170662

Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca2+ current in liver cells

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1016/J.ABB.2010.01.004

Abstract: Uncouplers of mitochondrial oxidative phosphorylation, including carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and carbonilcyanide m-cholorophenylhydrazone (CCCP), are widely used in experimental research to investigate the role of mitochondria in cellular function. Unfortunately, it is very difficult to interpret the results obtained in intact cells using FCCP and CCCP, as these agents not only inhibit mitochondrial potential, but may also affect membrane potential and cell volume. Here we show by whole-cell patch cl ing that in primary rat hepatocytes and H4IIE liver cells, FCCP induced large proton currents across the plasma membrane, but did not activate any other observable conductance. In intact hepatocytes FCCP inhibits thapsigargin-activated store-operated Ca(2+) entry, but in patch cl ing under the conditions of strong Ca(2+) buffering it has no effect on store-operated Ca(2+) current (I(SOC)). These results indicate that there is no direct connection between mitochondria and activation of I(SOC) in liver cells and support the notion of indirect regulation of I(SOC) by mitochondrial Ca(2+) buffering.

The glucagon-like peptide-1 analogue exendin-4 reverses impaired intracellular Ca 2+ signalling in steatotic hepatocytes

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.BBAMCR.2016.05.006

Abstract: The release of Ca(2+) from the endoplasmic reticulum (ER) and subsequent replenishment of ER Ca(2+) by Ca(2+) entry through store-operated Ca(2+) channels (SOCE) play critical roles in the regulation of liver metabolism by adrenaline, glucagon and other hormones. Both ER Ca(2+) release and Ca(2+) entry are severely inhibited in steatotic hepatocytes. Exendin-4, a slowly-metabolised glucagon-like peptide-1 (GLP-1) analogue, is known to reduce liver glucose output and liver lipid, but the mechanisms involved are not well understood. The aim of this study was to determine whether exendin-4 alters intracellular Ca(2+) homeostasis in steatotic hepatocytes, and to evaluate the mechanisms involved. Exendin-4 completely reversed lipid-induced inhibition of SOCE in steatotic liver cells, but did not reverse lipid-induced inhibition of ER Ca(2+) release. The action of exendin-4 on Ca(2+) entry was rapid in onset and was mimicked by GLP-1 or dibutyryl cyclic AMP. In steatotic liver cells, exendin-4 caused a rapid decrease in lipid (half time 6.5min), inhibited the accumulation of lipid in liver cells incubated in the presence of palmitate plus the SOCE inhibitor BTP-2, and enhanced the formation of cyclic AMP. Hormone-stimulated accumulation of extracellular glucose in glycogen replete steatotic liver cells was inhibited compared to that in non-steatotic cells, and this effect of lipid was reversed by exendin-4. It is concluded that, in steatotic hepatocytes, exendin-4 reverses the lipid-induced inhibition of SOCE leading to restoration of hormone-regulated cytoplasmic Ca(2+) signalling. The mechanism may involve GLP-1 receptors, cyclic AMP, lipolysis, decreased diacylglycerol and decreased activity of protein kinase C.

Using continuous porous silicon gradients to study the influence of surface topography on the behaviour of neuroblastoma cells

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1016/J.YEXCR.2007.10.015

Abstract: The effects of surface topography on cell behaviour are the subject of intense research in cell biology. These effects have so far only been studied using substrate surfaces of discretely different topography. In this paper, we present a new approach to characterise cell growth on porous silicon gradients displaying pore sizes from several thousands to a few nanometers. This widely applicable format has the potential to significantly reduce s le numbers and hence analysis time and cost. Our gradient format was applied here to the culture of neuroblastoma cells in order to determine the effects of topography on cell growth parameters. Cell viability, morphology, length and area were characterised by fluorescence and scanning electron microscopy. We observed a dramatic influence of changes in surface topography on the density and morphology of adherent neuroblastoma cells. For ex le, pore size regimes where cell attachment is strongly discouraged were identified providing cues for the design of low-fouling surfaces. On pore size regimes more conducive to cell attachment, lateral cell-cell interactions crosslinked the cell layer to the substratum surface, while direct substrate-cell interactions were scarce. Finally, our study revealed that cells were sensitive to nanoscale surface topography with feature sizes of <20 nm.

Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.REDOX.2015.11.001

STORE‐OPERATED Ca²⁺ CHANNELS AND MICRODOMAINS OF Ca²⁺ IN LIVER CELLS

Publisher: Wiley

Date: 2009

DOI: 10.1111/J.1440-1681.2008.05095.X

Abstract: 1. Oscillatory increases in the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) play essential roles in the hormonal regulation of liver cells. Increases in [Ca(2+)](cyt) require Ca(2+) release from the endoplasmic reticulum (ER) and Ca(2+) entry across the plasma membrane. 2. Store-operated Ca(2+) channels (SOCs), activated by a decrease in Ca(2+) in the ER lumen, are responsible for maintaining adequate ER Ca(2+). Experiments using patch-cl recording and the fluorescent Ca(2+) reporter fura-2 indicate there is only one type of SOC in rat liver cells. These SOCs have a high selectivity for Ca(2+) and properties essentially indistinguishable from those of Ca(2+) release-activated Ca(2+) (CRAC) channels. 3. Although Orai1, a CRAC channel pore protein, and stromal interaction molecule 1 (STIM1), a CRAC channel Ca(2+) sensor, are components of liver cell SOCs, the mechanism of activation of SOCs, and in particular the role of subregions of the ER, are not well understood. 4. Recent experiments have used the transient receptor potential vanilloid 1 (TRPV1) non-selective cation channel, ectopically expressed in liver cells, and a choleretic bile acid to deplete Ca(2+) from different ER subregions. The results of these studies have provided evidence that only a small component of the ER is required for STIM1 redistribution and the activation of SOCs. 5. It is concluded that different Ca(2+) microdomains in the ER and cytoplasmic space are important in both the activation of SOCs and in the signalling actions of Ca(2+) in liver cells. Future experiments will investigate the nature of these microdomains further.

Expression of Drosophila Ca²⁺ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival

Publisher: Springer Science and Business Media LLC

Date: 21-07-2003

DOI: 10.1038/SJ.CGT.7700608

Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis

Publisher: Portland Press Ltd.

Date: 15-06-2002

DOI: 10.1042/BJ20020061

Abstract: Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the α- and β-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92kDa or 120kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated β-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.

Linkage Projects - Grant ID: LP0562583

Start Date: 03-2006

End Date: 09-2009

Amount: $136,444.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0347705

Start Date: 2003

End Date: 12-2006

Amount: $84,099.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0210511

Start Date: 2002

End Date: 12-2004

Amount: $140,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0559562

Start Date: 2005

End Date: 12-2008

Amount: $260,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP140100259

Start Date: 06-2014

End Date: 06-2018

Amount: $390,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0210512

Start Date: 2002

End Date: 12-2006

Amount: $470,299.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0347962

Start Date: 2002

End Date: 12-2003

Amount: $218,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0561115

Start Date: 2005

End Date: 12-2006

Amount: $474,500.00

Funder: Australian Research Council