Immunoblotting Protocol for Protein Analysis

Immunoblotting was performed as described previously^{32 (link)}. Briefly, total protein lysates were prepared using a radio-immunoprecipitation assay (RIPA; Beijing Dingguo Changsheng Biotechnology, Beijing, China) lysis buffer supplemented with the protease inhibitor phenylmethanesulfonyl fluoride (PMSF; Beijing Dingguo Changsheng Biotechnology, Beijing, China). Protein concentrations were determined using a BCA kit (Beijing Dingguo Changsheng Biotechnology, Beijing, China). Proteins were separated on SDS-PAGE gels, and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). Then, the membranes were probed overnight at 4°C with the following primary antibodies: KCTD-12, CD271, E-cadherin, N-cadherin and β-actin. Next, the membranes were washed with Tris Buffered Saline Tween (TBST), and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Beijing Dingguo Changsheng Biotechnology, Beijing, China). Protein bands were visualized with hypersensitive chemiluminescence kit (Wanleibio, Shenyang, China). Immunodetection was accomplished using the ChemiDoc XRS system (Bio-Rad, USA). Finally, densitometric analyses were performed using ImageLab software (Bio-Rad, USA).