Pgex 4t2 vector

SPANXA was PCR-amplified from pcDNA3-SPANXA-FLAG generated as described above, and subcloned into pGex-4T2 vector (Addgene). One liter of cultured BL21(DE3)pLysS competent cells (Promega) previously transformed by electroporation was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at 0.4 mM for 4 h at RT. Cells were washed twice with ice-cold PBS and centrifuged for 10 min at 5000 g. Cells were lysed in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 20 mM β-mercaptoethanol and 1X EDTA-free protease inhibitor cocktail (Merck), sonicated and centrifuged 20 min at 14,000 g at 4° C. The supernatant was incubated with 5 mL of gluthatione sepharose beads (GE Healthcare Life Sciences), pre-washed twice with lysis buffer overnight at 4° C. Beads were then washed 5 times with Hepes 25 mM pH 7.6. Elution of SPANXA was performed by on-beads digestion at RT for 9 h with 100 units of thrombin. Samples were resolved by SDS-PAGE and stained with Coomassie blue (Biorad) according to the manufacturer’s intructions. The band corresponding to SPANXA was cut from the gel and used as antigen to immunize rabbits (ProSci Inc, CA). Bleeds obtained after 3–4 immunizations were used for Westerns and IP as noted.