Figure 5.

TrkA, GIPC, and GAIP form an immunoprecipitable protein complex. (A) GAIP (∼25 kDa) coprecipitates with GIPC (∼40 kDa). pcDNA3.1-mGIPC and pcDNA3-hGAIP were transiently cotransfected into HEK293 cells followed by metabolic labeling with [³⁵S]methionine. Cell lysate was immunoprecipitated (IP) with preimmune (lane 1) or anti-GIPC (lane 2) serum. (B) Duplicate coimmunoprecipitations were performed as in A followed by immunoblotting with either anti-GIPC (top) or anti-GAIP (bottom) to verify the identity of the 25 kDa band as GAIP. GAIP is detected in the immune (lane 2) but not the preimmune (lane 1) precipitate. (C) Coimmunoprecipitation of TrkA, GIPC, and GAIP. pCMV5-TrkA (lanes 2 and 4) or empty vector (lanes 1 and 3) were coexpressed with pcDNA3.1-mGIPC and pcDNA3-hGAIP in HEK293 cells. Lysates were immunoprecipitated with anti-Trk (C-14) (lanes 2 and 4) followed by immunoblotting with anti-Trk (44) (top), anti-GIPC (middle), and anti-GAIP (bottom) antisera. GIPC and GAIP coimmunoprecipitated with TrkA in cells transfected with all three proteins (lane 2) but not in the sample transfected with GIPC, GAIP, and empty vector (lane 1). The expression levels in 50 μg cell lysates are shown in lanes 3 and 4.