Alliance system software

Protein extracts were prepared as previously described [28 (link)]. Briefly, cells were lysed in [30 mM Tris pH 7.5; 100 mM NaCl; 10 mM MgCl2; 1% Triton X-100; 1 mM DTT; 0,5 mM Na3VO4; protease inhibitor cocktail (Sigma Aldrich)], briefly sonicated, incubated on ice for 20 min and centrifuged for 20 min at 12,000 g at 4 °C. Protein extracts were then analysed by Western Blot using the following primary antibodies: rabbit anti-HER3/ErbB3 (D22C5) (mAb#12,708, Cell Signaling), anti- AR (06–680, Millipore), anti-PCF11 (A303-706A, Bethyl Laboratories), anti-CSTF3 (A301-094, Bethyl Laboratories), anti-RBM39 (HPA001591, Atlas Antibodies), anti-H3 (Ab1791, Abcam), anti-H3K27me3 (mAb#9733, Cell Signaling); mouse anti-HSP90 (sc-13119, Santa Cruz), anti-ACTIN (sc-47778 Santa Cruz), anti-EZH2 (AC22) (mAb#3147, Cell Signaling), anti-MDM4 (clone 7A8, 04–1556, Sigma Aldrich). Anti-rabbit, anti-mouse (GE Healthcare) HRP-linked secondary antibodies were all used at 1:5000 dilution and ECL signal developed using Clarity Western ECL Substrates (Biorad) or Amersham ECL Select™. Densitometric analyses were performed using Alliance system software (UVITEC, Cambridge).

Total cell extracts were obtained by lysis in RIPA buffer as described [28 (link)]. Cellular fractionation was performed as described in [40 (link)]. Western Blot were performed as described [26 (link), 28 (link)] using following primary antibodies: mouse anti-hnRNP F/H (dilution 1:1000, Abcam), anti-VIMENTIN (dilution 1:2000, Sigma Aldrich), anti-TUBULIN (dilution 1:1000, Sigma Aldrich), anti-NEK2 (dilution 1:500, Santa Cruz), anti-NF-YA, anti-HSP90a/b, anti-GAPDH, anti-SRSF1 (dilution 1:1000, Santa Cruz); goat anti-MATRIN3 (1:500, Santa Cruz); rabbit anti-H3 (dilution 1:1000, Abcam) and anti-E-CADHERIN (1:1000, Cell Signalling). Densitometric analysis were performed using Alliance system software (UVITEC, Cambridge).