For immunoprecipitation, HeLa cells were washed with PBS and then solubilized for 30 min at 4°C in TNE buffer (10 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 0,2% NP-40) with proteinase inhibitor (complete, Roche). The lysate was centrifuged at 4°C for 20 min at 16,000 x g. The supernatant was pre-cleared with Protein-G sepharose 4B Fastflow (Protein-G, GE Healthcare) and then incubated at 4°C for 4 hr with the indicated antibody and Protein-G sepharose 4B Fastflow. Beads were washed three times with TNE buffer. Precipitated proteins were eluted by boiling in SDS-PAGE sample buffer and analyzed by immunoblotting with appropriate antibodies.
For GST pull-down assay, cell lysates prepared as described above were incubated with 10 μg GST fusion proteins for 1 hr at 4°C. After the addition of glutathione-Sepharose 4B (GE Healthcare), the samples were incubated for 1 hr at 4°C. The resins were washed three times with TNE buffer, and bound proteins were eluted with 20 mM glutathione, mixed with SDS-PAGE sample buffer and analyzed by immunoblotting with appropriate antibodies. IP3 (Dojindo) was added at the beginning of the experiment when stipulated. Pull-down assay using recombinant proteins was performed similarly in TNE buffer with 10 μg GST fusion proteins mixed with recombinant IRBIT (1 μg) and/or recombinant Bcl2l10 (1 µg).
For GST pull-down assay, cell lysates prepared as described above were incubated with 10 μg GST fusion proteins for 1 hr at 4°C. After the addition of glutathione-Sepharose 4B (GE Healthcare), the samples were incubated for 1 hr at 4°C. The resins were washed three times with TNE buffer, and bound proteins were eluted with 20 mM glutathione, mixed with SDS-PAGE sample buffer and analyzed by immunoblotting with appropriate antibodies. IP3 (Dojindo) was added at the beginning of the experiment when stipulated. Pull-down assay using recombinant proteins was performed similarly in TNE buffer with 10 μg GST fusion proteins mixed with recombinant IRBIT (1 μg) and/or recombinant Bcl2l10 (1 µg).