Restore stripping buffer

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).

Western blotting was performed as described previously^{30 (link), 31 (link)} with modifications. Briefly, cell layers were rinsed twice in PBS and lysed in ice-cold RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Sigma cat# P8340) and 3mM EDTA, with all steps performed on ice. 20 or 40µg of total protein per sample was separated on 8% SDS-PAGE gels and electroblotted overnight at 18V constant voltage onto PVDF membrane in transfer buffer (25mM Tris, 192mM glycine, 20% methanol, 0.01% SDS, pH 8.3). Membranes were blocked in TBS + 5% BSA/1% milk and incubated overnight at 4^oC with primary antibodies followed by washing and incubation with peroxidase conjugated secondary antibodies. Immunoreactive bands were visualized using the SuperSignal West Dura or Femto Chemiluminescence kits (ThermoFisher Scientific) and imaged on a Fujifilm LAS 4000 gel documentation system using the Multi-gauge software (Fujifilm, Tokyo, Japan). Blots were stripped using Restore stripping buffer (Thermo Fisher Scientific) and re-probed with other primary antibodies of interest then lastly probed with HRP-anti-β-actin to confirm equal protein loading. Densitometry was performed using the Multi-gauge software.