Anti p2 7 antibody

Cells were detached by scraping and lysed in lysis buffer (300 μM sucrose, 1 mM K₂HPO₄, 1 mM MgSO₄, 5.5 mM glucose, 20 mM HEPES (pH 7.4), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.2 μg DNase, and 0.3 μg RNase, all by Sigma-Aldrich) by repeated freeze/thawing cycles. Proteins were separated on 8% Bolt-SDS precast gels (Thermo Fisher Scientific) and blotted on nitrocellulose paper (GE Healthcare Life Science). Membranes were incubated overnight with the anti-P2×7 antibody (Sigma-Aldrich), at a dilution of 1:200 in Tris-buffered saline (TBS: 50 mM Tris, 150 mM NaCl, pH 7.6), supplemented with 3% non-fat milk and 0.5% bovine serum albumin (BSA). The anti-β actin Ab was used at a dilution of 1:1000 in TBS plus 5% BSA. The secondary anti-rabbit horseradish peroxidase-conjugated Ab (Dako, Milan, Italy) was used at a dilution of 1:2000 in TBS-t buffer (TBS, 0.1% Tween-20, Sigma-Aldrich). Protein bands were revealed with an enhanced chemiluminescence detection kit (GE Healthcare Life Science).

For western blotting, the following antibodies were used: anti-GFP, anti-tubulin, anti-Ub, anti-E2-25K, anti-synaptophysin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-caspase-3, anti-S5a antibodies (Cell Signaling, Beverly, MA, USA); anti-SUMO1, anti-NSE antibodies (Zymed, Carlsbad, CA, USA); anti-S10 antibody (Genetex, Irvine, CA, USA); anti-S5b antibody (Novus, Littleton, CO, USA); anti-S5a, anti-S7, anti-PAC2, anti-β5, anti-20S core antibodies (Biomol, Farmingdale, NY, USA); anti-S2 antibody (Abcam, Cambridge, UK); anti-p27 antibody (Sigma-Aldrich, St. Louis, MO, USA); and anti-S6a antibody (Enzo, Plymouth Meeting, MA, USA). Anti-E2-25K antibody was described previously.^{28 (link)}