Enhanced chemiluminescence reagent

Tumor tissues and cells were washed with PBS and underwent lysis with RIPA buffer (Pierce) supplemented with protease inhibitors (Boster Biological Technology, China). Protein quantitation utilized the BCA assay kit (Beyotime Biotechnology). Equal amounts of total protein were resolved by SDS-PAGE and underwent transfer onto polyvinylidene fluoride membranes (Millipore, USA). Western blot membranes underwent overnight incubation at 4 °C with anti-FAM126A (1:1000; Sigma, #84668), anti-ENO1 (1:1000; Proteintech, #11204), anti-GHPDH (1:1000; Proteintech, #60004), anti‑E‑cadherin (1:1000; Proteintech, #20874), anti-N-cadherin (1:1000; Proteintech, #22018), anti‑vimentin (1:1,000; Proteintech, #10366), anti-snail (1:1000; Proteintech, #13099), anti-cyclin D1 (1:1000; Cell Signaling Technology [CST], #55506), anti-cyclin E1 (1:1000; CST, #4136), anti-CDK 2 (1:1000; CST, #2561), anti-CDK4 (1:1,000; CST, #12790), anti-AKT (1:1000; CST, #4691), anti-p-AKT (1:2000; CST, #4060), anti-PI3K (1:1000; CST, #4249), anti-p-PI3K (1:1000; CST, #17366), anti-Ki-67(1:800; CST, #9449) and anti-PCNA (1:1000; CST, #2561) primary antibodies. This was followed by incubation with HRP-linked anti-rabbit IgG (Boster, #BA1041). Enhanced chemiluminescence reagent (Proteintech, #7003) was used to detect immunoreactive signals.

Total cell proteins were extracted in a mixture containing phosphatase inhibitor, protease inhibitor, and RIPA lysis buffer (Cwbio, Beijing, China) at a ratio of 98:1:1. The protein concentration was assessed using a BCA Assay Kit (Beyotime, Shanghai, China). A standard quantity of proteins was electrophoresed on SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated overnight with a primary antibody at 4 °C and then with a secondary antibody for 1 h. The bands were measured using an enhanced chemiluminescence reagent (Proteintech, Chicago, IL, USA) and an ECL system (General Electric Company, Boston, MA, USA). Each experiment was repeated in triplicate.

Anti-FAM63B (1:500; ThermoFisher, # 62318), Anti-ACTN4 (1:5,000; Proteintech, #66628), Anti-GHPDH (1:1,000; Proteintech, #60004), anti-E-calmodulin (1:1,000; Proteintech, #20874), anti-N-calmodulin (1:1000; Proteintech, #22018), anti-wavoprotein (1:1,000; Proteintech, #10366), anti-snail (1:1000; Proteintech, #13099), anti-cyclin D1 (1:1,000; Cell Signaling Technology [CST], #55506), anti-cyclin E1 (1:1,000; CST, #4136), anti-CDK 2 (1:1,000; CST, #2561), anti-CDK4 (1:1,000; CST, #12790), anti-AKT (1:1,000; CST, #4691), anti-p-AKT (1:2,000; CST, #4060), anti-PI3K (1:1,000; CST, #4249), anti-p-PI3K (1:1,000; CST, #17366), anti-mTOR (1:1,000; CST, #2972), anti-p-mTOR (1: 1000; CST, #2971), HRP-goat anti-rabbit IgG (Boster, #BA1055), HRP-goat anti-mouse IgG (Boster, #BA1050), Anti-PCNA (1:1,000; Proteintech, # 10205), Anti-Ki67 (1:1,000; Proteintech, #28074), HA-Ubiquitin plasmid (Sangon Biotech, China), CHX (Melun Biologics, China), MG132 (MCE, USA), MINDY2 small interfering RNA (RiboBio, China), protease inhibitor (Boster Biological Technology, China), and enhanced chemiluminescence reagent (Proteintech, #7003).