Cleaved caspase 3 ab2302

20 μg protein extract were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk and followed by incubating with the primary antibody (FUT4, AP12067b, Abgent; cleaved caspase-3 ab2302, Abcam; cleaved PARP, ab4830, Abcam; Sp1, ab13370, Abcam; CD44, ab157107, Abcam; GSK-3β, 22,104–1-AP, Proteintech; p-GSK-3β, 22,104–1-AP, Proteintech; β-catenin, 51,067–2-AP, Proteintech; CyclinD1, 60,186–1-Ig, Proteintech; GAPDH, AP7873a, Abgent) on a shaker overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (rabbit IgG, 1/1000 diluted; UK). A GAPDH antibody (1/200 diluted; Santa Cruz Biotech) was used as a control. All bands were detected using ECL Western blot kit (Amersham Biosciences, UK). The bands were measured with LabWorks (TM ver4.6, UVP, BioImaging systems).

Protein extraction and western blotting were performed as described previously [43 (link)]. Briefly, cells or tissues were lysed on ice for 30 min in radio-immunoprecipitation assay buffer. The lysates were centrifuged at 12,000 rpm at 4°C for 15 min, the supernatants were collected, and protein concentrations were determined using bicinchoninic acid assay (KenGEN, Jiangsu, China). Equal amounts of protein were separated by 10% SDS-PAGE followed by electrotransfer onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, MA, USA). Membranes were blocked with 5% nonfat milk for 2 h and incubated with primary antibodies. An electrochemiluminescence detection system (Thermo Fisher Scientific) was used for signal detection. Antibodies against p53(#2527), p21(#2947), bax(#5023), cyclinB1(#12231), cyclinE1(#4129), CDK2(#2546), DAPK1(#3008) and β-actin (#3700) were obtained from Cell Signaling Technology (MA, USA), and cleaved caspase 3(ab2302) obtained from Abcam.