Las 4000 mini analyzer

The cells were lysed in lysis buffer, and 20 µL of protein lysate sample was fractionated on polyacrylamide gels (TGX™ FastCast™ Acrylamide Kit; Bio-Rad Laboratories, Hercules, CA, USA) and then electroblotted to nitrocellulose membranes. The membranes were blocked with 5% skim milk in phosphate buffered saline-Tween 20 (PBS-T). The membranes were incubated with primary and then secondary antibodies. They were then treated with enhanced chemiluminescence detection reagents (Amersham™; Cytiva, Marlborough, MA, USA), and chemiluminescent signals were visualized as bands using a LAS 4000 mini analyzer (Cytiva).
Antibodies against phospho-cdc2 (Try15), cyclin D1, cyclin B1, cleaved caspase-3, caspase-3, phospho-AKT (Ser473), AKT, phospho-WNK1 (Thr60), WNK1, phospho-GSK-3β (Ser9), GSK-3β, phospho-mTOR (Ser2448), and mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Monoclonal beta-actin antibody (FUJIFILM Wako Pure Chemical Corp.) was used to probe an internal control.

Cells were dissolved in lysis buffer, and 10 µL of each protein sample was fractionated on polyacrylamide gels (TGX™ FastCast™ Acrylamide Kit; Bio-Rad Laboratories, Hercules, CA, USA) and then electroblotted onto nitrocellulose membranes. The membranes were blocked and incubated with primary antibodies against β-actin (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) or KLF4 (Novus Biologicals, Littleton, CO, USA), and then the membranes were incubated with horseradish peroxidase-conjugated secondary antibody. The membranes were then treated with enhanced chemiluminescence detection reagents (Amersham™; Cytiva, Marlborough, MA, USA), and chemiluminescent signals were visualized as bands using a LAS 4000 mini analyzer (Cytiva).