Clarity western substrate kit

Immunoblotting was conducted as previously described [25 (link), 75 (link)]. Specifically, samples with equal amounts of protein (~10 μg) were dissolved in Laemmli buffer containing 5% β-mercaptoethanol (Bio Rad, #1610737) and subjected to sodium dodecyl sulfate-polyacrilamide gel electrophoresis (8–16% gradient), using pre-cast gels (BioRad, #1610710; Hercules, CA), followed by protein transfer onto polyvinylidene difluoride membranes (BioRad, #1704159). The membranes were incubated in 5% blocking buffer (BioRad, #1706404) dissolved in TBST (100 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature, followed by an overnight incubation at 4°C with primary antibodies dissolved in blocking buffer. Membranes were then washed with TBST and incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Chemiluminescent technique using the Clarity Western Substrate kit (BioRad, #1705061) was employed to visualize protein bands in a ChemiDoc XRS + imaging system (BioRad). Membranes were subsequently stripped with Restore Stripping Buffer (ThermoFisher Scientific, #46430; Rockford, IL) and re-probed with anti-α-tubulin or anti-actin antibodies to serve as the loading control. Densitometric analysis of protein bands was performed with the ImageJ software (National Institutes of Health).