Western Blot Protein Analysis Protocol

Cell protein lysates were obtained as previously described [12 (link),28 (link)]. Protein levels were quantified with the BCA protein assay kit (Thermo Scientific, Rockford, IL). Protein (15 μg) was mixed with NuPAGE^® Reducing agent containing NuPAGE^® LDS sample buffer (Invitrogen, Carlsbad, CA), heated at 70°C for 10 min, and loaded into Novex^® 4–12% Bis-Tris precast gels (Invitrogen, Carlsbad, CA) for SDS-PAGE. Proteins were transferred onto 0.45 μm PVDF membrane and blocked with 5% BSA in TBST (20mM Tris-HCl, PH 7.6, 150mM NaCl, 0.1% Tween 20). Membranes were incubated with primary antibodies at 4°C overnight, and washed three times in TBST. Afterwards, secondary antibodies were applied at room temperature for 1 h, followed by three washes with TBST. Bands were visualized with Supersignal^® HRP substrate (Thermo Scientific, Rockford, IL) and imaged with Bio-Rad Gel Doc™ system (Bio-Rad, Hercules, CA).