Isopropyl thio β galactoside

Escherichia coli BL21 bacteria harboring pET28a-bcnA were grown for 3 h with 500 mM isopropyl thio-β-galactoside (ThermoFisher Scientific, United Kingdom) at 25°C; bacteria were harvested, resuspended in 50 mM phosphate buffer (PBS), pH 7.4 (Sigma-Aldrich, United Kingdom), with protease inhibitors (Roche Diagnostic GmBH, Germany), and lysed using a cell disruptor at 27 kpsi (Constant Systems). The lysate was cleared by centrifugation at 16,000 × g for 60 min at 4°C (Sorvall RC 6 Plus, Germany), and the supernatant was filtered through 0.2-μm in-line filters. The supernatant was passed through a HisTrap FF 5-ml column, using AKTA Start (GE Healthcare Bio-science, Sweden). The purified protein was detected by Coomassie blue staining following 16% SDS-PAGE and quantified by Bradford assay using bovine serum albumin (BSA) as standard.

6x-His Tag monoclonal primary antibody (His.H8, ThermoFisher), STIM1 monoclonal antibody (CDN3H4, ThermoFisher), mCherry polyclonal antibody (PA5-34974, ThermoFisher), GAPDH polyclonal antibody (G9545, SigmaAldrich), goat anti-mouse and anti-rabbit secondary antibodies DyLight 800 and DyLight 680 (ThermoFisher) were used for western blots. OxotremorineM (Sigma-Aldrich), thapsigargin (Invitrogen/Life-technologies), isopropylthio-β-galactoside (ThermoFisher), Fura-PE3 AM (Teflabs), Protease Inhibitor (PI) Cocktail Set 1 (Calbiochem/EMD Millipore), 0.01% poly-L-lysine solution (Sigma), Ni-IDA resin (Protino/Macherey-Nagel), Lipofectamine2000 (ThermoFisher). Following concentration of chemicals are used in all the experiments unless noted: OxotremorineM, 10 μM; thapsigargin, 1 μM; isopropylthio-β-galactoside, 0.5 μM. All lipids were obtained from Avanti Polar Lipids; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 850375C; 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine 16:0-18:1 (POPS), 840034C; 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 840035; L-α-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), 840046; L-a-phosphatidylinositol-4-phosphate (PI(4)P), 840045X; L-a-phosphatidylinositol (PI), 84042C.