G3bp2

Whole cell lysates were used for immunoblotting as previously described²² (link). Briefly, cells were washed with cold PBS, and then lysed with RIPA lysis buffer containing protease and phosphatase inhibitor. Floating cells or cell fragments were collected and lysed with the above lysates. Protein concentrations were determined by Bradford assay. Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Membranes were blocked in TBST containing 5% nonfat skim milk at room temperature for 2 h and probed with primary antibodies overnight at 4 °C. Then the membranes were blotted with an appropriate horseradish peroxidase-linked secondary antibody. Electochemiluminescence was performed according to the manufacturer׳s instructions with ChemiImager 5500 imaging system (Alpha Innotech Co., CA, USA). Antibodies against Akt, phospho-Akt (Ser423), ERK and phospho-ERK were from Cell Signaling Technology (Danvers, MA, USA). G3BP2 was from Abcam (Cambridge, MA, USA), and anti-β-actin was from Sigma (St Louis, MO, USA). Other anti-G3BP1, HRP-linked goat anti-rabbit and anti-mouse secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).