Additional file 5 of PD-1 blockade delays tumor growth by inhibiting an intrinsic SHP2/Ras/MAPK signalling in thyroid cancer cells

Additional file 5: Supplementary Figure 4. Effects of intrinsic PD-1 on SHP2 localization and functions. A. Expression levels of PD-1, phospho-PD-1, SHP2 and phospho-SHP2 in 8505c and TPC-1 cells transiently transfected with pFLAG PD-1 or the empty vector pFLAG, assessed by western blot. A representative experiment is shown. B. Immunofluorescence microscopy of 8505c cells, transiently transfected with pFLAG PD-1 or the empty vector, with antibody specific for SHP2. Arrows indicate the surface signal of SHP2. Bars, 5 μm. A representative experiment is shown. C. Immunofluorescence microscopy of 8505c cells transiently transfected with pCMV6 PD-1-GFP and stained with antibody specific for SHP2, and the merged signal. Arrows indicate the surface signal of SHP2. Bars, 5 μm. A representative experiment is shown. D. Total protein extracts from TPC-1 cells transiently transfected with pFLAG-PD-1 or the empty vector pFLAG were subjected to a pull-down assay using the indicated recombinant proteins or to immunoprecipitation using the indicated antibodies. Proteins were immunoblotted with antibody against SHP2 or GRB2. A representative experiment is shown. E. Total cell protein extracts from 8505c cells transiently transfected with combination of pCEFL H-Ras AU5, pFLAG PD-1 or empty vector (pFLAG + pCEFL) were subjected to immunoprecipitation with anti-phospho tyrosine followed by western blotting with pan (RAS) antibody. A representative experiment is shown, together with the mean densitometric analysis ± SD of 5 independent assays. * P