Anti ip3r

Reagents used were AZC (TCI Europe, Zwijndrecht, Belgium; # A1043 or Acros Organics, Geel, Belgium; # 105142500), bafilomycin A1 (Sanbio, Uden, The Netherlands; # 11038-500), ethylene glycol tetraacetic acid (Acros Organics; # 409910250), BAPTA-AM (Thermo Fisher Scientific, Waltham, MA, USA; # B6769), TG (Alomone labs, Jerusalem, Israel; # T-650), Fura-2 AM (Life Technologies, Carlsbad, CA, USA; # F1221), staurosporine (LC Labs, Woburn, MA, USA; # S9300), and AMG PERK 44 (Tocris, Abingdon, U.K.; # 5517).
Primary antibodies used were anti-ATF6 (Cell Signaling Technology, Leiden, The Netherlands; # 65880), anti-BiP (Cell Signaling Technologies; # 3183), anti-eIF2α (Cell Signaling Technology; # 9722), anti-phospho-eIF2α (Cell Signaling Technology; # 3398), anti-ERp57 (Cell Signaling Technology; # 2881), anti-ERp72 (Cell Signaling Technology; # 2798), anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA; # G8795), anti-IP₃R (Rbt475 [42 (link)] recognizing all IP₃R isoforms), anti-LC3 (Cell Signaling Technology; # 2775), anti-MCU (Sigma-Aldrich; # HPA016480), anti-PARP (Cell Signaling Technology; # 9532), anti-PMCA (Thermo Fisher Scientific; # MA3-914) recognizing all PMCA isoforms, anti-SERCA2B (Cell Signaling Technology; # 4435), and anti-vinculin (Sigma-Aldrich; # V-9131).

Homogenized liver tissues and samples were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). Quantified protein extracts (40 μg) were loaded in 6 ~ 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The following primary antibodies were used: anti-SERCA2b (1:500; Invitrogen), anti-mitochondrial calcium uniporter (MCU) (1:1000; Invitrogen), anti-IP3R (1:800; Cell Signaling Technology), anti-voltage-dependent anion-sensitive channel 1 (VDAC1), anti-total-eukaryotic initiation factor 2α (eIF2α), anti-phospho-eIF2α, anti-activating transcription factor 4 (ATF4), anti-CHOP, anti-PI3K-p110α (1:1000; all from Cell Signaling Technology), anti-PI3K-p85 (1:3000; BD Biosciences, San Jose, CA, USA), anti-PERK (1:200; Santa Cruz), anti-GRP75 (1:500; Abcam), and anti-calmodulin (1:500; Novus Biologicals). The loading control was anti-GAPDH (1:2000; AbFrontier, Seoul, Republic of Korea). After the membranes were washed, the following secondary antibodies were used: anti-mouse IgG and anti-rabbit IgG (1:8,000; all from Bio-Rad). The protein bands were detected using a Clarity Western ECL kit (Bio-Rad) and a ChemiDoc imaging system (Bio-Rad).

All DNA constructs were generated by a PCR-based method and sequenced to confirm their fidelity. Orai1 and STIM1 were amplified from Orai1 (MMM1013-20276444), and STIM1 (MMM1013-202764946) cDNA purchased from Open Biosystems and introduced into pEBB vectors. For Orai1-CFP and STIM1-YFP vector construction, CFP and YFP were amplified from Raichu-Rac1 [25 (link)] and C-terminally introduced into pEBB-Orai1 and pEBB-STIM1, respectively. Anti-Flag (Sigma, F1804, St. Louis, MO, USA), anti-Orai1 (Santa Cruz, sc-68895, Dallas, TX, USA), anti-Orai1 (Abcam, ab111960, Cambridge, UK), anti-STIM1 (Abcam, ab108994), anti-IP₃R (Cell Signaling, #8568, Boston, MA, USA), anti-phospho-IP₃R (Cell Signaling, #3760S), anti-PLCγ1 (Cell Signaling, 2822S), anti-phospho-PLCγ1 (Cell Signaling, 2821S), anti-Mer (R&D systems, AF591, Minneapolis, MN, USA), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.