Cell lysate preparation and Pin1 immunoblotting

Expression of Pin1, its corresponding variants, and PKCα levels were monitored by immunoblotting. Cells were solubilized in lysis buffer (BB150: 50 mM Tris pH 7.6, 150 mM NaCl, 0.2% CHAPS, 10 mM EDTA) on ice and centrifuged at 21,000×g for 5 min at 4°C to remove insoluble materials. Protein in the soluble fractions was quantified using the colorimetric bicinchoninic acid assay (Pierce) and samples were prepared with 80 µg of total lysate protein in 1× Laemlli buffer (62.5 mM Tris-HCl, 2% SDS, 25% glycerol, and 0.01% bromophenol blue, pH 6.8). Samples were subsequently boiled for 5 min and resolved by SDS-PAGE using 15% acrylamide gels (120 V constant voltage for 80 min) and transferred onto 0.2 µm nitrocellulose membranes (Bio-Rad) at 350 mA (constant current) for 1 hr. Following transfer, membranes were blocked for 1 hr in 5% milk powder (wt/vol) in 20 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween 20 (TBST), and incubated at 4°C overnight with primary antibodies at a 1:1000 dilution in Tris-buffered saline (pH 7.6), 0.1% Tween 20, 5% BSA (wt/vol) with rocking. Following incubation, blots were washed three times for 10 min each in TBST and incubated with goat anti-rabbit secondary antibody coupled to horseradish peroxidase (MillliporeSigma; 1:10,000 dilution) in TBST/5% BSA for 1 hr at room temperature. Following three additional 10 min washes with TBST, blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and analyzed by densitometry using a Molecular Imager ChemiDoc XRS+ system (Bio-Rad Laboratories, Hercules, CA, USA). Protein levels were normalized to total protein load and verified using actin as loading control.