Bp381 1

Lysates were loaded at 15 μg in Mini Protean 4–15% polyacrylamide gels (Bio Rad, 4508084). Electrophoresis was run at 120 V for 50 min in 1x tris-glycine-SDS running buffer [25 mM tris base (Sigma, T1503-1KG), 190 mM glycine (Fisher, BP381-1), 0.1% sodium dodecyl sulfate (Fisher, BP243-1)]. Proteins were transferred to Immobilon-P membranes (Millipore, IPVH85R) at 120V for 65 min at 4C in 1x tris-glycine-methanol transfer buffer [25 mM tris base, 190 mM glycine, 20% methanol (Fisher, A412P-4)]. Membranes were blocked with 5% milk (CAT) in 1x tris buffer saline with Tween-20 [TBST; 20 mM tris base, 150 mM Tween-20 (Sigma, P9416-100mL)] for 120 min at RT. Membranes were washed 3x with 1x PBS and incubated with appropriate primary antibody (Table 4) diluted in 4% BSA (Sigma, A9418-50G) in 1x PBS overnight at 4C. Membranes were washed 3x with 1x PBS and incubated with appropriate secondary antibody diluted in 1% BSA in 1x TBST for 60 min at RT. Membranes were washed 3x with 1x PBS and incubated with Clarity Max ECL substrate (Bio Rad, 1705061) for 5 min. Chemiluminescence images were obtained using the ChemiDoc imaging system (Bio Rad, 17001401). Volumetric band intensities were analyzed using Bio Rad Image Lab software.