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

Biochem J. 2002 Jun 15;364(Pt 3):641-8. 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. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Brain / metabolism
  • Calcium Channels / analysis*
  • Calcium Channels / genetics
  • DNA, Complementary
  • Guinea Pigs
  • Humans
  • Liver Neoplasms, Experimental
  • Molecular Sequence Data
  • Muscle, Smooth / metabolism*
  • Peptide Fragments / analysis
  • Peptide Fragments / chemistry
  • Protein Isoforms / analysis
  • Protein Isoforms / genetics
  • RNA, Messenger / genetics
  • Rats
  • Recombinant Proteins / analysis
  • Respiratory System / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • TRPC Cation Channels
  • Transcription, Genetic
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Calcium Channels
  • DNA, Complementary
  • Peptide Fragments
  • Protein Isoforms
  • RNA, Messenger
  • Recombinant Proteins
  • TRPC Cation Channels
  • transient receptor potential cation channel, subfamily C, member 1