Ab32531

Protein extraction was implemented utilizing RIPA Lysis Buffer (Beyotime). The concentrations of protein samples were examined with a BCA protein assay kit (Tiangen). After separation by 10% sodium dodecyl sulfonate‐polyacrylamide gel (SDS‐PAGE; Solarbio) electrophoresis, the protein sample (20 μg) was blotted onto polyvinylidene difluoride membrane (Corning) and then blocked with 5% non‐fat milk (Solarbio) for 1 h. Afterwards, the membranes were immunoblotted with primary antibodies against CHEK1 (1:10000, ab32531; Abcam), proliferating cell nuclear antigen (PCNA) (1:2000, ab152112; Abcam), Ki67 (1:1000, ab243878; Abcam), matrix metalloproteinase 9 (MMP9) (1:1000, ab38898; Abcam), Bcl‐2‐associated X protein (Bax, 1:1000, ab53154; Abcam) and GAPDH (1:10000, ab181602; Abcam) overnight at 4°C followed by interaction with Goat Anti‐Rabbit IgG H&L (HRP) secondary antibody (1:10000, ab205718; Abcam) for 2 h at indoor temperature. Finally, the intensity of protein bands was assessed utilizing BeyoECL Plus ECL Kit (Beyotime).

EC9706 cells were solubilized in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) with protease inhibitors (Roche, Basel, Switzerland). The protein concentrations were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA) according to the manufacturer’s instructions. Proteins (50 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific, Inc.). The membranes were then incubated with 5% milk–Tris-buffered saline-Tween (TBST) blocking buffer for 3 h at room temperature. After washing three times with PBS, the membranes were incubated with primary antibodies of B-cell lymphoma 2 (Bcl-2; ab59348), Bcl-2-associated X (Bax; ab53154), caspase 3 (ab32531), caspase 9 (ab32539), CXCR4 (ab124824), Wnt3a (ab28472), Wnt5a (ab72583), β-catenin (ab32572), phosphorylated inhibitor of NF-κB (p-IκBα; ab92700), total (t)-IκBα (ab32518), p-p65 (ab86299), t-p65 (ab32536), and GAPDH (ab181602; Abcam, Cambridge, UK) at 4°C overnight, subsequently added horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab205718; 1:5,000; Abcam), and incubated at room temperature for 1 h. An enhanced chemiluminescent kit (Thermo Fisher Scientific, Inc.) was then used to conduct chemiluminescent detection.