Phosphatase

Cell lysates for Western blots were made in RIPA buffer (Sigma) supplemented with protease (Roche) and phosphatase (Santa Cruz Biotechnology) inhibitor cocktails. We used the NE-PER® Nuclear and Cytoplasmic extraction reagents (Pierce Biotechnology) for cellular fractionation. For IHC, after deparaffinization and rehydration, tissue sections were antigen-retrieved at 95°C for 30 minutes. Immunostaining with anti-c-MET (Cell Signaling Technology) was performed using a streptavidin–biotin, horseradish peroxidase and DAB chromogen (Vector Labs). IHC with anti-YAP1 (Santa Cruz Biotechnology) and anti-LEF1 (Santa Cruz Biotechnology) was performed using alkaline phosphatase, vulcan fast red chromogen (Biocare Medical), and hematoxylin counterstain (Thermo Scientific). Immunostaning with anti-CD8 (Dako) and anti-CD163 (Abcam) was visualized by TRITC- and FITC-labeled secondary antibodies, respectively, and nuclei were counterstained by DAPI.

Cell lysates were made in RIPA buffer (Sigma) for direct Western blotting or in a PNE buffer (PBS:H₂O at 1 :1, 0.5% NP40, 5 mM EDTA, 5% glycerol) for immunoprecipitation, with both buffers supplemented with protease (Roche) and phosphatase (Santa Cruz Biotechnology) inhibitor cocktails. Western blots and immunoprecipitations were performed using the following antibodies : p-ERK1/2 (T202/Y204), p-MEK1/2 (S217/221), p-AKT (Thr308), p-CRAF (S338), total ERK1/2, MEK1/2, MEK1, MEK2, AKT, CRAF, DUSP4 and HA (Cell Signaling Technology), TUBULIN and FLAG (Sigma), BRAF (F-7), BRAF (C-19), p-MEK1 (Thr291), p-MEK1 (S222) (Santa Cruz Biotechnology) and p-MEK2 (S226) (US Biological). Western blot quantification was performed using NIH Image J. The three-dimension structures of MEK1 (3EQC) and PTEN mutants were modeled by the I-TASSER online server. Modeling the ^V600EBRAF-^MUTMEK1 dimer interface was based on the crystal structure of the ^WTBRAF-^WTMEK1 dimer (4MNE), the MEK1-KSR2 dimer (2Y4I), and the asymmetric, vemurafenib-bound, ^V600EBRAF dimer (3GO7). Protein structures were visualized using Pymol^™