Rabbit monoclonal anti snail c15d3

Western blot analysis was carried out as described elsewhere [49 (link)]. The following antibodies were used: rabbit polyclonal anti-β-tubulin (H-235) and mouse monoclonal anti-fibronectin (IST-9) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-Integrin α5 (AB1949) and goat polyclonal anti-HGF from R&D System (Minneapolis, MN, USA); rabbit polyclonal anti-RSK1, rabbit monoclonal anti-RSK2 (D21B2) XP, rabbit monoclonal anti-phospho-p90RSK (380), rabbit monoclonal anti-phospho-RSK2 (227) (D5EA11), rabbit polyclonal anti-YB1 (D299), rabbit monoclonal anti-phospho-YB1 (Ser102) (C34A2) and rabbit monoclonal anti-SNAIL (C15D3) obtained from Cell Signaling Technology (Beverly, MA, USA); mouse monoclonal anti-Vinculin (hVIN-1) from Sigma, Saint Louis, MO, USA. It is worth noting that two bands of YB-1 become visible when the level of YB-1 expression is very high as in the case of rescued cells. Indeed, two bands are recognized by several antibodies and are differently interpreted [see e.g. ref. 52 (link)].
Bound antibodies were detected using the appropriate peroxidase-conjugated secondary antibody and revealed by enhanced chemiluminescence (Pierce, Rockford, IL, USA).

The cells were lysed using extraction buffer (Thermo Scientific) and measured for protein concentration with a BCA protein assay kit (Pierce). The cell lysates were separated by 8–15% SDS-PAGE and transferred to a nitrocellulose (NC) membrane (Pall Corporation). Membranes were blocked with 5% skim milk in Tris-buffered saline/Tween 20 (TBS-T) buffer for 1 h at RT. After washing steps with TBS-T buffer, NC membranes were incubated with rabbit monoclonal anti-α-SMA (E184, 1:1000), rabbit polyclonal anti-HSD11B1 (1:500), rabbit polyclonal anti-N Cadherin (1:1000), mouse monoclonal anti-Vimentin (RV202, 1:1000) (all from Abcam), rabbit monoclonal anti-phospho-Smad3 (Ser423/425) (C25A9, 1:500), rabbit monoclonal anti-SMAD2/3 (1:1000), rabbit monoclonal anti-Snail (C15D3, 1:1000) (all from Cell Signaling Technology), rabbit polyclonal anti-Collagen I (Novus Biologicals, 1:500), and mouse monoclonal anti-β-actin (Sigma-Aldrich, 1:3000) for 16 h at 4 °C. After washing step, with TBS-T buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, 1:5000), and the specific bands were visualized by enhanced chemiluminescence (ECL; Thermo Scientific).