Ab62364

Total cellular proteins were extracted using the cell lysis buffer for Western blot. The protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Bio-Rad, USA). The membranes were blocked in 5% skim milk and then incubated with a specific primary antibody and a secondary antibody, and they were then detected by enhanced chemiluminescence (ECL). The immunoblots were visualized using the Image Quant LAS 4000 digital imaging system (GE, USA). The following primary antibodies were used: Antibodies for PHLPP2 (PA5-25995) and Vimentin (PA5-2723) were obtained from Thermo Fisher. Antibodies for GSK-3β (ab131356), p-GSK-3β (ab75814), P27 (ab62364), P21 (ab109520), CyclinD1 (ab134175), E-cadherin (ab152102) and Snail (ab82846) were purchased from Abcam. Antibodies for AKT (D260001) and p-AKT (D155022) were purchased from Sangon Biotech . While the β-actin antibody and the secondary antibodies were purchased from Beyotime.

Commercially available antibodies were as follows: CACYBP (11745-1-AP, Proteintech, 1:2000 for WB, 1:1000 for IF and 1:400 for IHC), Tubulin (66031-1-Ig, Proteintech, 1:5000 for WB), GAPDH (60004-1-Ig, Proteintech, 1:2000 for WB), Lamin B1 (66095-1-Ig, Proteintech, 1:1000 for WB), anti-Flag (F3165, Sigma, 1:3000 for WB and 1:1000 for IF), anti-HA tag (3724, Cell Signaling Technology, 1:1000 for WB and 1:500 for IF), anti-Myc tag (2276, Cell Signaling Technology, 1:1000 for WB), RNF41 (GTX115366, GeneTex, 1:1000 for WB and 1:200 for IHC), β-catenin (8480, Cell Signaling Technology, 1:1000 for WB), P27^Kip1 (610241, BD Biosciences, 1:1000 for WB and 1:500 for IF), phosphor-Ser10-P27^Kip1 (ab62364, Abcam, 1:1000 for WB), phosphor-Thr157-P27^Kip1 (AF1555, R&D Systems, 1:1000 for WB), phosphor-Thr198-P27^Kip1 (AF3994, R&D Systems, 1:1000 for WB), cyclin D1 (2978, Cell Signaling Technology, 1:1000 for WB), cyclin A2 (4656, Cell Signaling Technology, 1:1000 for WB), CDK2 (2546, Cell Signaling Technology, 1:1000 for WB), CDK4 (12790, Cell Signaling Technology, 1:1000 for WB). Small molecule inhibitors were as follows: MG-132 (474790, Calbiochem), chloroquine (S4157, Selleck), 3-methyladenine (3-MA, M9281, Sigma-Aldrich), cycloheximide (CHX, C7698, Sigma-Aldrich). All other chemical reagents were obtained from Sigma-Aldrich, unless otherwise indicated.