Anti arf6

Total protein was extracted from collected cells in RIPA buffer (Sigma-Aldrich; Merck KGaA) containing 1 mM PMSF (Sigma-Aldrich; Merck KGaA) and total protein concentration was quantified using a BCA assay (Beyotime Institute of Biotechnology). In total, 20 µg proteins were separated by 10% SDS-PAGE and subsequently transferred onto a PVDF membrane (EMD Millipore), which was blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) diluted in TBS-0.1% Tween-20 buffer at room temperature for 2 h. The membranes were then incubated with the following primary antibodies (all 1:1,000) at 4˚C overnight: Anti-ARF6 (Abcam; cat. no. ab226389), anti-Rac1 (Abcam; cat. no. ab155938), anti-Bcl-2 (Abcam; cat. no. ab194583), anti-Bax (Abcam; cat. no. ab263897), anti-Erk1/2 (Abcam; cat. no. ab17942), anti-p-ERK1/2 (Abcam; cat. no. ab214362) and anti-β-actin (Abcam; cat. no. ab213262). Following the primary antibody incubation, the membranes were washed with TBST and incubated with a HRP-conjugated secondary antibody (Abcam; cat. no. ab6721; 1:5,000) at room temperature for 2 h. Protein bands were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.; cat. no. 34080) on a VersaDoc™ gel imaging system (Bio-Rad Laboratories, Inc.). Densitometric analysis was performed using the Image J software (version 1.51; National Institutes of Health).