G coated sepharose beads

Cells (fibroblasts cell cultures or HEK-293, as indicated in the Figures) were collected and lysed in buffer containing 30 mM Tris-HCl, at pH 7.5, 50 mM NaCl, 1% NP-40. To map the BAP1 and IP3R3 binding region, HEK-293 cells were transiently transfected using polyethylenimine, collected 24 hours later, and lysed in 50 mM Tris, at pH 7.5, 150 mM NaCl, glycerol 10%, 1 mM EDTA, 50 mM NaF and NP-40 0.1%. All the buffers were supplemented with proteases and phosphatases inhibitors. Extracted proteins were pre-cleared by incubating lysates with G-coated sepharose beads (GE Healthcare) for 1 hour at 4 °C, then the supernatant (700 μg, referred as “Input”) was incubated overnight with IP3R3 antibody at 4 °C; precipitation of the immune complexes was performed with G-coated sepharose beads for 4 hours at 4 °C, according to manufacturer's instructions. Alternatively, the supernatant was incubated for 3 hours at 4 °C with FLAG^® M2 resin (Sigma, cat. no A2220) or HA resin (Roche, cat. no. 11815016001). After immunoprecipitation, the beads were washed three times with lysis buffer, at 4 °C, and suspended in 40 μl of 2X Laemmli buffer. 10-20 μl (depending on experiment) were loaded on the gel and the samples were processed by SDS-PAGE and analyzed by WB.