Western Blot Analysis of Endocytic Proteins

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and the cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in PBS with non-fat milk and probed with an anti-CHC antibody (1:1,000; ab21679, Abcam), anti-dynamin II antibody (1:1,000; ab151555, Abcam), anti-caveolin-1 antibody (1:1,000; C4490, Sigma), anti-Rab5 antibody (1:1,000; ab18211, Abcam), or an anti-Rab7 antibody (1:1,000; ab126712, Abcam) for 1 h at room temperature and subsequently washed three times with 0.1% Tween 20/PBS, followed by an incubation for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:10,000; BA1054, Boster) or HRP-conjugated goat anti-mouse secondary antibody (1:10,000; BA1050, Boster). The membranes were washed three times with 0.1% Tween 20/PBS and visualized by exposure to FluorChem HD2 Imaging System (Alpha Innotech) after the addition of a chemiluminescent substrate (SuperSignal West Dura Extended Duration Substrate; 34075; Thermo Scientific Pierce) (Li M. et al., 2017 (link)).