Snail

Cells lysis and western blot were carried out as previously described [18 (link)]. Antibodies for: ILK, Akt, E-cadherin, Vimentin, GSK-3β, phospho-GSK-3β (Y216), β-catenin (all Transduction Laboratories BD), AR, Src, phospho-Src (Y416), phospho-ERK (T202/Y204), phospho-Akt (S473), phospho-GSK-3β (S9), D₁, D₃ and phospho-eIF4E (S209) (all Cell Signaling Technology Inc.), N-cadherin (R&D) and β-actin, ZEB1, (all Sigma) SNAIL (ABGENT), Calnexin, HSP-90 (all Calbiochem) were used to detect indicated proteins. The presence of the primary antibody was revealed with horseradish peroxidase-conjugated secondary antibodies diluted 1:2000 (Cell Signaling Technology Inc) and visualized with an enhanced chemiluminescence detection system (Bio-Rad) as previously described [19 (link)]. β-Actin served as a loading control. All immunoblots were stripped with stripping buffer containing 25 mM glycine–HCl, pH 2, 1% (wt/v) SDS for 30 min, and incubated in antibody against β-actin (dilution, 1:3000; Sigma-Aldrich), which served as a loading control. To obtain quantitative results, immunoblots were scanned using the public domain ImageJ software (National Institute of Health). Each data point was normalized against its corresponding actin data point.

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF) and proteins (20–40 μg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies (1:1000), HRP-conjugated secondary antibodies (1:5000), and visualized in Imager (Bio-Rad) using ECL system (Thermo Fisher Scientific, Rochester, NY). Antibodies of ATM, p-ATM, JAK1, p-JAK1, JAK2, p-JAK2, STAT3, and p-STAT3, were from Gene Tex (Irvine, CA), Antibodies of E-cadherin, N-cadherin, Vimentin, Snail, Zeb1, Twist and VEGF were obtained from Abgent (San Diego, CA) and antibodies of PD-L1, GAPDH, were from Cell Signaling (Danvers, MA).