Anti cleaved caspase 3

Western blotting was performed using a dioctanoic acid protein detection kit (Cat #PC0020; Solarbio). Samples (40 to 60 µg/10 µL) were separated using 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyvinylidene difluoride membranes were subsequently used for protein transfer, and samples were incubated overnight at 4 °C with the following primary antibodies: anti-p-Akt (Ser473) (1:2000; RRID: AB_2315049; Cell Signaling Technology), anti-Akt (1:1000; RRID: AB_915783; Cell Signaling Technology), anti-p-mTOR (1:2000; Cat #R25033; ZenBio), anti-mTOR (1:2000; Cat #380,411; ZenBio), anti-p62 (1:2000; RRID: AB_10694431; Proteintech), anti-LC3 (1:1000; RRID: AB_2137737; Proteintech), anti-Bax (1:10 000; RRID: AB_2061561; Proteintech, Planegg-Martinsried Germany), anti-Bcl-2 (1:1000, Proteintech, RRID: AB_2227948), anti-cleaved caspase 3 (1:1000; Cat #341,034; ZenBio), anti-caspase 3 (1:1000; RRID: AB_331439; Cell Signaling Technology), anti-caspase 9 (1:500; RRID: AB_2068632; Proteintech), and anti-β-actin (1:5000; Cat #CL594-66009; Proteintech). Goat anti-mouse or anti-rabbit IgG secondary antibodies were incubated with horseradish peroxide (1:500; RRID: AB_449890; Abcam, Cambridge, UK). Signals were detected using a bioimaging system (ChemiDoc XRS+; Bio-Rad), and ImageJ software was used to analyze the band intensity.

A western blot assay was performed using protein extracted from cultured cells and tissues. Protein samples were separated using 10% and 12% SDS-PAGE and subsequently transferred onto PVDF membranes with a pore size of 0.22μm. The membrane was blocked using 5% skim milk and a primary antibody was then incubated overnight at 4 °C on the membranes. The primary antibodies were used as follows: anti-CENPW (1:1,000, 1030382-3; Abcam, United Kingdom), anti-βactin (1:10,000; 60004-1-Ig; Proteintech, Wuhan, China), anti-Cleavedcaspase 3 (1:1,000, #9661S; Zenbio, Chengdu, China), anti-CyclinB1 (1:1,000, 60186-1-Ig; Proteintech, Wuhan, China), anti-CDK1 (1:1,000, 11554-1-AP; Proteintech, Wuhan, China), anti-Bcl-2 (1:1,000, #2872T; CST, United States), anti-P21 (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China), anti-Bax (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China), and anti-STAT3 (1:1,000, #9139; CST, United States), anti-p-STAT3 (Tyr705) (1:2000, #9145; CST, United States). A chemiluminescence system was employed to detect protein bands subsequent to the incubation of primary antibody probes with secondary antibodies. The scanned blots were subjected to analysis using ImageJ version 1.51 in order to ascertain the density of the bands. Protein expression was normalized by comparing it to the expression of β-actin.