Western Blot Analysis of Cytoskeletal Proteins

Cells were harvested, and total cell protein lysates were prepared as previously described (Xu et al., 2011 (link)). In brief, cells were collected, washed with ice-cold PBS, resuspended in SDS PAGE sample buffer, and incubated at 100°C for 10 min. Protein concentrations were measured directly in the samples using NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Proteins were separated on 4–20% gradient Tris-Glycine polyacrylamide gels (Invitrogen), transferred onto PVDF membranes (Immubilon-P; Millipore), and incubated with primary antibodies, followed by HRP-conjugated anti–mouse (Roche) or anti–rabbit (Roche) secondary antibodies. Blots were detected with ECL PLUS reagent (GE Healthcare) using ImageQuant LAS4000 chemiluminescence imager (GE Healthcare). Antibodies used for Western blot were: anti-cortactin mAb (clone 4F11; Millipore), anti-cofilin polyclonal antibody (clone FL-166; Santa Cruz Biotechnology, Inc.), anti-Arp2 polyclonal antibody (clone H-84; Santa Cruz Biotechnology, Inc.), anti-ARC/p34 polyclonal antibody (Millipore), anti-Tom20 polyclonal antibody (clone FL-145; Santa Cruz Biotechnology, Inc.), anti-Opa1 mAb (clone 18; BD), and anti-Mfn2 mAb (Abcam).