Hrp conjugated mouse anti beta actin

Western blotting was performed as described previously (47 (link)). In short, total cell lysates were harvested in protein sample buffer (0.1 M Tris [pH 6.8], 4% SDS, 4 mM EDTA, 286 mM 2-mercaptoethanol, 3.2 M glycerol, 0.05% bromophenol blue), and proteins were resolved by SDS-PAGE. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes, probed with primary antibody diluted in PBS–0.1% Tween 20 (PBST) containing 5% skim milk overnight at 4°C, and then stained with secondary antibody diluted in PBST with 5% skim milk for 1 h at room temperature (RT). Probed proteins were detected using ECL reagents on a ChemiDoc system with Image Lab software (Bio-Rad). The following antibodies were used: mouse anti-HA (Frank W. Fitch Monoclonal Antibody Facility, The University of Chicago, clone 12CA5), goat anti-mouse CD300LF (R&D systems, AF2788), horseradish peroxidase (HRP)-conjugated mouse anti-beta-actin (Santa Cruz Biotechnology, sc-47778), and HRP-conjugated goat anti-mouse (BioLegend, number 405306).