Ab167389

LoVo or SW1116 cells were lysed on ice with the lysis buffer (Beyotime, Shanghai, China) and the lysates were later centrifuged for 15 min at 12,000 × g. The BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) was acquired for evaluating protein concentrations. Protein was subjected to SDS–PAGE (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Primary antibodies and secondary antibodies were applied to incubate membranes in sequence. ECL detection system (Applied Biosystems, Foster City, CA, USA) was utilized to visualize protein bands. Primary antibodies against MMP2 (ab97779, Abcam, Cambridge, MA, USA), MMP7 (ab5706, Abcam), N-cadherin (ab76057, Abcam), E-cadherin (ab40772, Abcam), NANOG (ab80892, Abcam), OCT4 (ab181557, Abcam), Gli4 (AV37797, Sigma-Aldrich), PTCH1 (ab53715, Abcam), Shh (ab53281, Abcam), Gli1 (ab49314, Abcam), Gli2 (ab167389, Abcam), and GAPDH (ab9484, Abcam) were used, individually.

Western blotting was performed using standard protocols. Briefly, cells were sonicated in RIPA buffer, and 30 μg proteins was resolved on 12% polyacrylamide SDS gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA (bovine serum albumin) in TBS (tris‐buffered saline), incubated overnight at 4°C with the appropriate primary antibodies. The antibodies used included those against VASH2 (ab116640, Abcam, Cambridge, UK), GAPDH (AG019‐1, Beyotime, Shanghai, China), Vimentin (ab8069, Abcam), E‐cadherin (ab11512, Abcam), ZEB1 (ab124512, Abcam), ZEB2 (ab25837, Abcam), Bcl‐2 (ab32124, Abcam), SMO (ab38686,, Abcam), Gli‐1 (ab49314, Abcam), and Gli‐2 (ab167389, Abcam).