For verification of knockdown experiments, cells were lysed in RIPA buffer 48 h after transfection. Extracted proteins were separated by 4–20% SDS-PAGE (Thermo Fisher Scientific), transferred to PVDF membranes (Bio-Rad Laboratories), incubated at 75 V for 2 h, and blocked for 1 h with 5% nonfat milk (Bio-Rad Laboratories) or BSA (Sigma-Aldrich). The PVDF membranes were incubated overnight at 4°C with appropriate antibodies: anti-ARF1 (dilution 1:1,000; ab108347; Abcam); anti-ARF6 (dilution 1:1,000; ab77581; Abcam); anti-ARNO (dilution 1:1,000; ab56510; Abcam); anti–cytohesin 1 (dilution 1:500; MABT14; EMD Millipore); anti–α-tubulin (dilution 1:3,000; T6199; Sigma-Aldrich); anti-βCOP (dilution 1:1,000; ab2899; Abcam); anti-HA (dilution 1:1,000; 2367; Cell Signaling Technology); anti-Cdc42 (dilution 1:1,000; 2462; Cell Signaling Technology); anti-RhoA (dilution 1:1,000; sc-418; Santa Cruz Biotechnology, Inc.); anti-Rac1 (dilution 1:1,000; 610650; BD); and anti–NM myosin-IIA (dilution 1:1,000; M8064; Sigma-Aldrich).
After three washes (10 min each), appropriate secondary antibodies conjugated with HRP (Bio-Rad Laboratories) were incubated with the membrane for 1 h, washed three times (15 min at RT), and detected by ECL Western blotting substratum (Thermo Fisher Scientific) using CL-Xposure film (Thermo Fisher Scientific).
After three washes (10 min each), appropriate secondary antibodies conjugated with HRP (Bio-Rad Laboratories) were incubated with the membrane for 1 h, washed three times (15 min at RT), and detected by ECL Western blotting substratum (Thermo Fisher Scientific) using CL-Xposure film (Thermo Fisher Scientific).