Anti pets1

Human MDA-MB-231 breast cancer cells were cultivated in monolayers in DMEM medium (Sigma Aldrich) supplemented with 10% foetal bovine serum (Clontech), penicillin and streptomycin (Sigma Aldrich). ERK1/2 inhibitors SCH772984 and VTX-11e were added at 100 nM final concentration for 4 hours, followed by two washes in PBS and release into fresh media for 0.5, 1 and 2 hours. Cells were harvested by trypsinization, washed once with cold PBS, re-suspended in SDS-PAGE loading buffer and sonicated. Equal amounts of protein were analysed by gel electrophoresis followed by Western blotting. NuPAGE-Novex 10% Bis-Tris gels (Invitrogen) were run according to manufacturer’s instructions. The following antibodies were used in immunoblotting: anti-pERK (Cell Signaling Technology), anti-pRSK (Abcam) and anti-pETS1 (Immunoway). Anti-ERK1/2 (Cell Signaling Technology), anti-GAPDH (Novus Biologicals) or anti-α-tubulin (Cancer Research UK Monoclonal Antibody Service) antibodies were used as loading controls.