Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis.
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. 80 kDa 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. 92 kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the alpha- and beta-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92 kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120 kDa 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 92 kDa or 120 kDa 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 beta-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.
Ong
Hwei Ling
HL
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia.
Chen
Jinglong
J
Chataway
Tim
T
Brereton
Helen
H
Zhang
Lei
L
Downs
Tamyra
T
Tsiokas
Leonidas
L
Barritt
Greg
G
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis* / Q000235:genetics; D018076:DNA, Complementary; D006168:Guinea Pigs; D006801:Humans; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D012137:Respiratory System / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D018076:DNA, Complementary; D010446:Peptide Fragments; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1