Western Blot Protocol for Protein Analysis

Proteins in cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis following a standard protocol and transferred to Immobilon polyvinylidene membranes (EMD Millipore, Burlington, MA, USA). Membranes were blocked with 5% milk in TBST for 1 h, followed by incubation with primary antibody (diluted in blocking buffer or 2% bovine serum albumin in TBST) overnight. Membranes were then washed three times in TBST, re-incubated with horseradish peroxidase-labeled secondary antibodies for 1 h, washed three times in TBST, and exposed to autoradiography films. Signals were detected by chemiluminescence (Thermo Fisher Scientific). Antibodies for CYTSA, α-tubulin, acetylated α-tubulin, and actin were obtained from Santa Cruz Biotechnology. All antibodies were used according to the manufacturers’ specifications. All cell lysates were prepared in RIPA buffer with protease and phosphatase inhibitors, as described previously [15 (link)]. The whole western blot figures can be found in supplementary materials.