Pierce enhanced chemiluminescence ecl plus western blotting substrate

Protein samples (20 μg/lane) were separated on 10% SDS-PAGE for 60 min at 80 V and transferred to a polyvinylidene difluoride membrane for 60 min at 80 V. The membrane was washed once in PBS with 0.2% Tween 20 (PBST) and then blocked with 5% non-fat milk for 1 h at room temperature (RT). The membrane was further probed with anti-MAP3K9 antibody (cat. no. ab228752), anti-β actin antibody (cat. no. ab115777), anti-matrix metalloproteinase (MMP)-9 antibody (cat. no. ab76003), anti-tissue inhibitor of metalloproteinase 1 (TIMP2) antibody (cat. no. ab180630), anti-epithelial (E)-Cadherin antibody (cat.no. ab40772), anti-vimentin antibody (cat. no. ab92547; all 1:1,000; Abcam) in non-fat milk and incubated overnight at 4°C. The membranes were then washed 3 times in PBST for 5 min and horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibodies (cat. no. ab205718; Abcam) were added at 1:2,000 dilution in non-fat milk for 1 h at RT. The membranes were then exposed to Pierce™ enhanced chemiluminescence (ECL) plus western blotting substrate (Thermo Fisher Scientific, Inc.) for 1 min in the dark. The resulting bands were scanned and the images were captured in the ChemiDoc MP (Bio-Rad Laboratories Inc.) and the gray level of the bands was analyzed by the ImageJ software (version 1.46; National Institutes of Health).