Figure 2
From: hTID-1 defines a novel regulator of c-Met Receptor signaling in renal cell carcinomas
The J domain of hTid-1 selectively interacts with kinase-inactive MetR. (a) A schema depicting the GST constructs used in the GST pull-down experiments: full-length Tid-1 construct, a J-domain construct, a G/F/Cys-rich and C-terminal construct. (b) 786-0 RCC cells were serum-starved and either left untreated (−) or treated with 40ng/ml HGF (+). Lysates were incubated with the indicated GST proteins and the resulting complexes were subjected to immunoblotting with specific antibodies to MetR, and a phosphotyrosine specific antibody (pTyr 4G10). To ensure equal amounts of GST proteins used, the SDS–PAGE gel was stained with Coomassie blue (bottom panel). (c) 786-0 RCC cells were serum-starved and either left untreated (−) or treated with HGF (+). Cell lysates were immunoprecipitated with Met or hTid-1 antibodies and immune complexes were resolved by SDS-PAGE and analyzed by western blotting using antibodies specific to phospho-Met (Tyr1234/1235 in activation loop, pMet), total Met and hTid-1. (d) Cell lysates processed as shown in panel c were immunoprecipated with pMet antibodies and immune complexes probed with Tid-1 and pMet antibodies. (e) 786-0 Tid1 null cells were infected with adenoviruses expressing either Ad-GFP, Ad-hTid-1S or Ad-hTid-1L and stimulated with HGF as indicated. Cell lysates were immunoprecipitated with Met antibodies and immunoblotted with either total Met, Tid-1 or pMet antibodies.
