Fig. 4

Effects of intrinsic PD-1 on SHP2 localization and functions. a. Total cell protein extracts from 8505c and TPC-1 cells transiently transfected with pFLAG PD-1 or the empty vector (pFLAG) were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. A representative experiment is shown. b. Total protein extracts from 8505c and TPC-1 cells transiently transfected with pFLAG-PD-1 were subjected to an in vitro pull-down assay using the indicated recombinant proteins. Bound proteins were immunoblotted with antibody against PD-1. A representative experiment is shown. c. 8505c cells transiently transfected with PD-1 (pFLAG PD-1) or stably overexpressing PD-1 (pCMV3 PD-1 cl16) and the relative control cells were harvested and subjected to cell protein fractionation. Membrane (M) and cytoplasmic (C) protein fractions were immunoblotted with the indicated antibodies. Transferrin receptor or tubulin levels were used as normalization of membrane and cytosolic fractions, respectively. A representative experiment is shown. d. Immunofluorescence microscopy of TPC-1 cells, transiently transfected with pFLAG PD-1 or the empty vector, stained with an antibody specific for SHP2. A representative experiment is shown. Arrows indicate the surface signal of SHP2. Bars, 5 μm. e. SHP2 phosphatase activity assay on TPC-1 and 8505c cells transiently transfected with PD-1 (pFLAG PD-1) and the relative control (pFLAG), assessed by using a specific SHP2 phosphorylated substrate in the presence of the Malachite Green tracer, a colorimetric method (absorbance at 620 nm) for the detection of free inorganic phosphate. The SHP2 phosphatase activity was normalized for SHP2 content as assessed by western blot. Data are presented as mean ± SD of 3 independent experiments. f. Total cell protein extracts from 8505c cells transiently transfected with pCEFL H-Ras AU5 + pFLAG PD-1 or empty vector (pFLAG) were subjected to immunoprecipitation followed by western blotting with the indicated antibodies. A representative experiment is shown, together with the mean densitometric analysis ± SD of 5 independent assays. * P < 0.05 compared to the relative control