Anti snail 1 antibody

HaCaT cells were grown to confluence on 24-well microplates (Iwaki) and scratched with or without the combination of 0.1 μg/ml poly I:C and anti-IL-8 antibody (1:200) as described above. Cells were fixed with 4% paraformaldehyde 24 h after the scratch. Cells were washed with 1% bovine serum albumin (BSA, Sigma) in PBS and incubated with 3% BSA for 30 min. Next, the cells were incubated with anti-IL-8 antibody (1:50; IBL), anti-TGF-β1 antibody (1:100; Cell Signaling Technology), anti-E-cadherin antibody (1:100; Abcam, Cambridge, UK), anti-vimentin antibody (1:150; Abcam), and anti-Snail 1 antibody (1:150; Abcam) for two hours at room temperature. Then, the cells were washed and incubated with the secondary antibody, CF488A-labeled anti-rabbit antibody (Biotium, Hayward, CA, USA), for 30 min at room temperature. For negative controls, the primary antibody was replaced with 1% BSA in PBS. Samples were counterstained with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (Biotium) for nuclear staining. To maintain cytokines in the cells, brefeldin A (50 μg/ml; Sigma) was added to the culture medium 4.5 h before fixation (only for IL-8 and TGF-β1 staining).

Western blot was performed as described previously [48 (link)]. The blots were incubated overnight with primary anti-PARP-1 antibody (Abcam), anti-CXCL1 antibody (Abcam), anti-MMP9 antibody (Abcam), anti-VEGFA antibody (Abcam), anti-E-cardherin antibody (Abcam), anti-Vimentin antibody (Abcam), anti-Snail1 antibody (Abcam), anti-Slug antibody (Abcam), anti-Twist antibody (Abcam), and anti-GAPDH antibody (Abcam).

RIPA buffer (Sigma Aldrich, Cambridge, MA) was used to lyse the cells to obtain total protein lysates. Protein concentration was measured using the BCA method (Sigma Aldrich). The quantified protein (25 μg) was transferred onto polyvinylidene fluoride (PVDF) membranes following SDS-PAGE gel electrophoresis. Then, the membrane was blocked with 5% nonfat dry milk in tri-buffered saline plus Tween (TBS-T) buffer for 2 h at room temperature and incubated with respective primary antibodies (1:1000 dilution) at 4 °C overnight, followed by Horseradish peroxidase-conjugated (HRP) secondary antibody (1:5000, Abcam, cat. no. ab7090) at room temperature for 1 h. The following primary antibodies were used: anti-HER-2 antibody (Abcam, cat. no. ab227383), anti-E-cadherin antibody (Abcam, cat. no. ab186533), anti-Snail-1 antibody (Abcam, cat. no. ab8614), anti-N-cadherin antibody (Abcam, cat. no. ab182651), anti-vimentin antibody (Abcam, cat. no. ab8805), anti-β-catenin antibody (Abcam, cat. no. ab8932), anti-GAPDH antibody (Invitrogen, cat. no. PA1–987).