Western Blot Analysis of EMT Markers

As described by Chen et al and his colleagues [25 (link)], after 48 h of transfection, cells were harvested for protein extraction. Proteins were quantified with a BCA protein assay kit (Solarbio). Then, an aliquot of 20 μg protein was electrophoresed on SurePAGE gels (Beyotime) and transferred onto nitrocellulose membranes using XCell II Blot Module (Thermo Fisher). The membranes were subjected to incubation with the antibodies for N-cadherin (1:500; Thermo Fisher), E-cadherin (1:1000; Thermo Fisher), SPARCL1 (1:1000; Thermo Fisher), and β-actin (1:3000; Affinity, Nanjing, China) after being blocked with 5% skimmed milk (Solarbio). The nitrocellulose membranes were incubated with secondary antibodies (1:3000; Affinity). At last, a Bio-Rad image analysis system was performed to develop protein blots with eyoECL Plus (Beyotime). The protein expression was normalized to β-actin.