Anti orai1

Cellular and tissue proteins were extracted with lysis buffer, which contained 1% (vol/vol) Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 8.0, with the addition of Roche protease inhibitor cocktail tablets (Sigma–Aldrich, St. Louis, MO, USA, 04693132001). The protein concentrations were determined with the DC Protein Assay (Bio-Rad, Hercules, CA, USA, 500-0002). The proteins were separated in 12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA in TBS for 2 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibody was anti-GAPDH (1:5000, Abcam, Cambridge, UK, ab8245), anti-Orai1 (1:1000, Sigma–Aldrich, St. Louis, MO, USA, O8264), anti-STIM1 (1:1000, Cell Signaling, Danvers, MA, USA 5668S), anti-ANF (1:3000, Abcam, ab14348), or anti-cTnT (1:3000, Abcam, ab8295). Immunodetection was accomplished using horseradish peroxidase-conjugated secondary antibody, followed by analysis via the ECL detection system. The intensity of immunoblot bands was quantified using ImageJ Software.

Proteins were extracted, quantified and separated as previously described¹⁶ . The primary antibodies used were: anti-Kv10.1 (1:200, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), anti-Orai1 (1:200, Sigma Aldrich, Saint-Quentin-Fallavier, France), anti-SPCA2 (1:250, Santa Cruz Biotechnology, Inc.), anti-ERK1/2 (1:500, Cell Signaling Tech., Danvers, USA), anti-p-ERK1/2 Thr202/Tyr204 (1:500, Cell Signaling Tech.). GAPDH (1:1,000, Cell Signaling Tech.) antibody was used for loading control experiments. Detection and quantification were realized as previously described¹⁶ .

Total protein was extracted with lysis buffer having 1% (vol/vol) Nonidet P-40, 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), and Roche protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, United States, 04693132001). The sample protein concentrations were determined using the DC Protein Assay (Bio-Rad, Hercules, CA, United States, 500–0002). The protein samples were resolved by 12% SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% BSA in TBS for 2 h at room temperature (RT), and then overnight incubated with primary antibodies at 4°C. The used primary antibodies were as follows: anti-GAPDH (1:6000, Abcam, Cambridge, United Kingdom, ab8245), anti-Orai1 (1:1000, Sigma–Aldrich, St. Louis, MO, United States, O8264), anti-STIM1 (1:1000, Abcam, ab108994), anti-ANF (1:1000, Abcam, ab14348), anti-β-Actin (1:1000, Abcam, ab14348), and anti-LC3 (1:1000, SIGMA, ab8295). Immunoreactive bands were visualized using the horseradish peroxidase-conjugated secondary antibodies. The intensity of immunoblotted bands was quantified using the ImageJ software.

Cells were lysed in protein lysis buffer (50 mM Tris, 100 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemented with 1x protease and phosphatase inhibitors (Roche Applied Science, Penzberg, Germany, 11836153001 and 04906845001). Gel electrophoresis was performed using Mini-PROTEAN^® TGX Pre-cast Gels, and protein was transferred to a PVDF membrane (Bio-Rad Laboratories, Hong Kong, China, 4568084 and 1704157). Membranes were blocked for 1 h in 5% skim milk in phosphate-buffered saline containing 0.1% Tween-20 (Sigma-Aldrich, St. Louis, MI, USA, P9416) (PBST) before incubating overnight at 4 °C with anti-ORAI1 (Sigma-Aldrich, St. Louis, MI, USA #08264 1:4000) or anti-vinculin (Cell Signaling, Danvers, MA, USA # 13901, 1:1000). Goat anti-rabbit horseradish peroxidase conjugate secondary antibodies (Bio-Rad Laboratories, Hong Kong, China, 1706515) were diluted 1:10,000 in 5% skim milk in PBST and incubated for 1 h at room temperature. Proteins were imaged using the SuperSignal West Dura Extended Duration Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA, 34076) on the Bio-Rad ChemiDoc Imaging System (Bio-Rad Laboratories, Hong Kong, China).

Source of specific antibodies: anti-αB-crystallin (mouse, 1:2000), anti-Hsp 20 (rabbit,1:10,000), anti-S16 Hsp-20 (rabbit, 1:500), anti-Hsp27 (rabbit, 1:2000), anti-S82 Hsp27 (rabbit, 1:2000), anti-S59 αB-crystallin (rabbit, 1:2000) and anti-S45 αB-crystallin (rabbit, 1:1000) from Abcam; anti GRP78 (rabbit, 1:500), anti-CASQ2 (rabbit, 1:2000) and anti-CRT (rabbit, 1:1000) from Thermo; anti Bcl-2 (rabbit, 1:1000) and anti-CNX (rabbit, 1:1000) from Santa Cruz; anti-Actin (rabbit, 1:1000), anti-STIM1 (rabbit, 1:2000), anti-TRPC3 (rabbit, 1:500), anti-Orai1 (rabbit, 1:500), anti-EDEM1 (rabbit, 1:1000) and anti-Herp (rabbit, 1:500) from Sigma; anti-Beclin1 (rabbit, 1:5000) and anti-HRD1 (1:1000) from Novus Biologicals; anti-GRP94 (rabbit, 1:1000) from Stressgen; mono- and polyubiquitinated conjugates (mouse, 1:1000) from EnzoLifeScience. Anti Derlin-1 (mouse, 1:1000) was a generous gift of Doriana Sandonà.

Sequence-specific gRNA for 5′-UTR Orai1 (5′-GTG​AGG​CCG​GGC​CCG​CGT​AGG​GG-3′) was designed using an online tool (http://crispr.dbcls.jp/) and subcloned into pX459 (pSpCas9(BB)-2A-Puro V2.0; Addgene plasmid 62988) using the BbsI recognition site. Jurkat E6.1 T cells were transfected with the generated CRISPR plasmids using Nucleofector 4D electroporation kit SF (Lonza) according to the manufacturer’s instructions. Cells were then cultured for 72 h after transfection in medium containing 1 µg/μl puromycin to select transfected cells. A monoclonal cell line was generated by infinite dilution, and the efficiency of protein deletion was evaluated by Western blot analysis. Immunoblots were probed with anti-Orai1 (O8264; Sigma-Aldrich).

For protein expression analysis, cells were harvested, washed with PBS then lysed in buffer containing 20 mM Tris, 100 mM KCl, 10% glycerine (v/v), 0.5% n-Dodecyl beta Maltoside (DDM), pH 7.4. Lysates were stored at −80° until analyzed. Standard SDS-PAGE was performed followed by electrotransfer to nitrocellulose membranes. Immunoblots were probed with anti-Orai1 (Sigma, catalogue number O8264), anti-STIM1 (Proteintech, # 11565-1-A), anti-STIM2 (Sigma, # S8572), anti-GAPDH (Cell Signaling, clone 14C10) anti-ß-actin (Abcam, clone AC-15) or anti-PMCA4 (Thermoscientific, clone JA9) at a 1:1,000 dilution. For protein detection, an enhanced chemiluminescence detection reagent was used (Clarity Western ECL Substrate, Biorad). Densitometric quantification of detected protein bands was done with Quantity one software (Biorad).