Ab105861

Total protein was extracted from cells and tissues, and the protein concentrations were measured using a BCA kit (Thermo Fisher Scientific, USA). The proteins were then separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose (NC) membrane. The membranes were blocked with Tris-buffered saline-Tween (TBST) solution containing 5% skim milk. Next, the membranes were incubated with anti-Cldn7 antibody (ab27487, 1:1000; Abcam), anti-Sox9 antibody (ab185230, Abcam), anti-Olfm4 antibody (ab105861, Abcam), anti-Ki67 antibody (ab16667, Abcam), anti-β-catenin antibody (ab32572, Abcam), anti-cyclin D1 antibody (ab134175, Abcam), anti-C-myc antibody (ab32072, Abcam), anti-E-cadherin antibody (ab40772, Abcam), anti-Snail-1 antibody (ab180714, Abcam) or anti-vimentin antibody (ab8978, Abcam) at 4 °C overnight, followed by an incubation with a donkey anti-rabbit IgG antibody (ab175780; Abcam) or anti-goat IgG antibody (ab175780; Abcam). Finally, a Western blot scanner was used to visualize the blots. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the cytoplasmic internal reference, and lamin B1 was used as the nuclear internal reference.

Total proteins from cultured cells were lysed in the RIPA buffer (Beyotime, China) and quantified. After SDS‐PAGE assay, we transferred the proteins onto polyvinylidene fluoride (PVDF) membranes followed by 5% nonfat milk blocked for 1 hour (h). Next, we incubated the membranes with primary antibodies overnight at 4 °C and then washed using phosphate buffered saline supplemented with Tween 20 (PBST). We subsequently incubated the membranes with secondary antibodies at room temperature for 2 h. In the final, we took the protein bands on membranes into visualization using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, USA). The used primary and secondary antibodies were listed as follows: rabbit anti-OLFM4 antibody (1:2000, Abcam, ab105861), rabbit anti- IGF2BP3 antibody (HRP) (1:1500, Abcam, ab208869), rabbit anti-CLDN1 antibody (1:2000, Abcam, ab180158), rabbit anti-MMP1 antibody (1:2000, Abcam, ab38929), rabbit anti-GAPDH (1:3000, Abcam, ab181603) and goat anti-rabbit IgG H&L (HRP) (1:3000, Abcam, ab205718). We used GAPDH as the endogenous control.