Flotillin 2 b6

Carbenoxolone (CBX), BAPTA-AM and thapsigargin (TG) were purchased from Tocris Bioscience (Ellisville, MO). APT102, the recombinant form of soluble CD39, was kindly provided by Dr. Ridong Chen at APT Therapeutics Inc. (St. Louis, MO). All other chemicals and cell culture media were from Sigma-Aldrich (St. Louis, MO) and other culture reagents from Life Technologies (Carlsbad, CA), unless otherwise stated.
Rabbit anti-mouse P2X7 antibody (#APR-004) was obtained from Alomone labs (Jerusalem, Israel); beta-actin (AC-15, #ab6276) from Abcam (Cambridge, MA); Flotillin-2 (B6, #sc-28320) from Santa Cruz Biotechnology, Inc. (Dallas, TX); Caveolin-1 (7C8, #NB100-615) from Novus Biologicals (Littleton, CO); phospho-AKT (S473) (#9271), phospho-PRAS40 (T246) (#2997), phospho-S6K (T389) (#9205), phospho-S6 (S235/236) (#2211) from Cell Signaling Technology (Danvers, MA); HRP-conjugated goat anti-mouse (#31438), donkey anti-rabbit (#31458) and mouse anti-goat IgG (#31400) and the SuperSignal West Femto Maximum Sensitivity Substrate reagents (#PI-34096) were from Thermo Scientific (Rockford, IL).

Total membrane protein extracts, FTs and eluates were analyzed by SDS-PAGE. Ten μg of proteins from each sample and 3 μL of BenchMark Protein Ladder (or 10 μL of BenchMark Prestained Protein Ladder for western blot, WB) were loaded on gels (Mini-PROTEAN TGX gels, 4–15%, BIO-RAD, United States). The dried eluates were suspended in 20 μL of 1x Laëmmli buffer and loaded totally on the gel. Proteins were stained with Coomassie Brilliant Blue R250 and gels were destained so as to obtain the optimum contrast.
The WB analyses were performed on PVDF membranes (transfer 1 h at 100 V in Tris-Glycine buffer inside a Mini Trans-Blot Electrophoretic Transfer cell) against adenylosuccinate lyase (ADSL C-11 at 1/500), α-adducin (C-4, 1/1000) and flotillin-2 (B-6, 1/6000) from Santa Cruz Biotechnology, United States.