Anti gst b 14

GST-fused protein of mouse VDAC1 was expressed in XL-10 cells using the pGEX6P-1 plasmid vector (GE Healthcare) and then purified by reduced glutathione-sepharose beads (GE Healthcare). His 6 -Flag-tagged mouse RNF207 proteins including deletion mutants were expressed in Rosetta blue cells with the use of the pET30 plasmid vector (Novagen, Madison, WI) and then purified with the use of ProBond metal affinity beads (Invitrogen).
For the production of recombinant proteins in Sf9 cells, we subcloned full-length mouse Rnf207 cDNAs into pFastBac HTc with epitope tags and expressed epitope-tagged full-length mouse Rnf207 with the BAC-to-BAC system (Clontech Laboratories, Mountain View, CA). Baculovirus infections, culture and affinity purifications were performed as described previously [29] . In vitro binding assays were performed as described previously [30] 2.9. Antibodies
We used anti-FLAG M2 antibodies (Sigma), anti-HA antibodies (Covance, Princeton, NJ), anti-VDAC1 antibodies (ab14734, Abcam, Cambridge, UK), anti-GST (B-14, Santa Cruz Biotechnology, Santa Cruz, CA), anti-PARP, anti-GAPDH (Cell Signaling Technology [CST], Danvers, MA) and anti-HSP90 (BD Transduction Laboratories, San Diego, CA).

EZH2-HIS 201-252 and EZH2 with individual mutations were immobilized on the His-Pur Cobalt resin and allowed to interact overnight with GST-VAV1-172 or GST alone (control) in PBS buffer pH 7.4. Following this, the beads were extensively washed and subject to SDS-PAGE and Western blot analysis using anti-His (h-3, Santa Cruz) and anti-GST (B-14, Santa Cruz) antibodies from Santa Cruz to analyze the EZH2-VAV complex. To determine the interaction between full-length EZH2 and VAV, VAV1 protein was immunoprecipitated from the cytosolic fraction of Jurkat cells with α-VAV1 antibody from Santa Cruz (C-14) and immobilized on Protein G Sepharose. Similarly, Flag-tagged EZH2 WT or EZH2 with individual mutations were immunoprecipitated from HEK-293 cells with α-Flag antibody from Sigma (M2) and Protein G Sepharose beads. They were then eluted from the beads by using a competing FLAG peptide. Equal quantities of the WT EZH2 or EZH2 with individual mutations were incubated overnight with VAV1 in a BC-100 buffer. The beads were then extensively washed and analysed by Western blotting with α-EZH2 antibodies from Santa Cruz (N-20) and VAV-specific antibodies.