Ibright 1500

Western blotting was performed as described previously (17 (link)). Briefly, control and Orai shRNA transduced LAD2 cells (1 × 10⁶) were lysed in radioimmunoprecipitation assay buffer (1× RIPA) with protease inhibitor cocktail, and the protein concentration was measured by the BCA protein assay. Twenty five micrograms of protein samples were applied to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to PVDF membrane. After brief blocking (5% skim milk, 1 h), blots were incubated with antibodies against Orai1 (1:500), Orai2 (1:500), Orai3 (1:500) and β-Actin (1:1,000) at 4°C overnight. Blots were incubation with specific horseradish peroxidase (HRP)-conjugated secondary antibodies for another hour, followed by incubation with an HRP substrate for ECL and image captured on iBRIGHT 1500 (ThermoFisher, Waltham, MA, USA). Signal quantitation was carried out after normalization to β-Actin loading controls, as indicated. For the assay with Orai inhibitor, LAD2 cells (2 × 10⁶) were preincubated with Synta66 (10 µM) for 30 min, stimulated with SP (1 µM) for different time intervals (0, 5, 15 and 30 min) and cell lysates were prepared. Protein samples were run in SDS-PAGE, incubated with anti-phospho-pERK1/2 (1:2,000), anti-phospho-Akt (1:1,000), anti-ERK1/2 (1:2,000), and anti-Akt (1:1,000) antibodies and processed similarly.

Cells were harvested and lysed with protease inhibitor-containing EBC buffer (50 mM Tris pH 8.0, 120 mM NaCl, 0.5% NP-40). After lysing, cells were centrifuged at 4 °C. The resulting supernatant was removed prior to quantification of protein concentration using Bradford Assay. Laemmli buffer and β-Mercaptoethanol was added to equal amounts of protein in cell lysates. Protein samples of 100 – 200 μg were subsequently separated with 12% and 8% Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis before transfer to PVDF membrane (Bio-Rad). Primary antibodies were added overnight following blocking in 5% milk in PBS-T, and were as follows: rabbit His-tag antibody (Cell Signalling Technology, RRID:AB_2115720), mouse CHD antibody (Santa Cruz Biotechnology, RRID:AB_10610044), mouse monoclonal p16INK4a antibody (BD Pharmingen, RRID:AB_394077), mouse monoclonal β-actin (Santa Cruz Biotechnology, RRID:AB_2833259), mouse FLAG-tag antibody (Sigma-Aldrich) and rabbit cleaved caspase-3 (CC3) antibody (Cell Signalling Technology). Visualisation was conducted using iBright1500 (Thermo Fisher). Bands were analysed using ImageJ software and normalized to β-actin.

Cytoplasm and membrane fractions were mixed with 4x Laemmli and β-mercaptoethanol. Equal volumes of fractions were directly loaded in a 4-12% pre-cast gradient gel (BioRad, USA) and separated by SDS-Page. Proteins were transferred to a nitrocellulose membrane (BioRad, USA), blocked with 5% non-fat dietary milk (NFDM, Carl Roth, Germany), and washed and incubated with the primary antibodies were either diluted in 5% NFDM or 5% bovine serum albumin (BSA, Serva, USA) overnight at 4°C: hIL-1α (1:500, sc-271618, clone G10, Santa Cruz, USA), mIL-1α (1:1,000, AF-400-SP, R&D, USA), GAPDH (1:5,000, sc-47724, clone 0411, Santa Cruz, USA), NaK ATPase (1:10,000, ab76020, Abcam, United Kingdom). The membrane was incubated with the secondary antibody coupled with horse radish peroxidase diluted in 5% NFDM for 1h on the following day. Classico Western HRP Substrate (Millipore) or SuperSigna West Femto (Thermo Fisher Scientific, USA) were used for development on iBright 1500 (Thermo Fisher Scientific, USA).

Western blotting was performed as previously described [31 (link)]. Briefly, equal mass aliquots of isolated cytoplasmic, nuclear, and input fractions were diluted with H₂O to 7.5 μl, mixed with 2.5 μl of NuPAGE LDS buffer (Invitrogen, catalog # NP0007), and heated to 70 °C for 10 min for denaturation. The isolates were separated on a NuPAGE 4–12% Bis-tris gel and transferred to a PVDF membrane. The primary antibodies used were mouse anti-tubulin alpha antibody (AA43) developed by Walsh, C (obtained from the Developmental Studies Hybridoma Bank at the University of Iowa, Department of Biology) and rabbit anti-histone H3 antibody (Novus Biologicals, catalog # NB500–171). The secondary antibodies used were goat anti-mouse IgG (H + L) antibody conjugated to HRP (Invitrogen, catalog # 31430) and goat anti-rabbit IgG (H + L) antibody conjugated to HRP (Promega, catalog # W4018). Immunoblots were imaged using iBright 1500 (ThermoFisher, catalog # A44241).

Fluorescein (5′-FAM) labeled oligonucleotides (0.1 μM) dissolved in 10 mM Tris–HCl (pH 8.0) and 0.5 μM EDTA buffer were heated at 95°C for 5 min and subsequently cooled to room temperature. Next, 100 mM LiCl, 100 mM KCl or 0.5 μM GRPC/100 mM KCl was added to the oligonucleotides, and the mixtures were immediately treated with 5% DMS reagent (Innochem, China) for 2 min on ice. Reactions were terminated by adding 80 μl stop buffer (3 M sodium acetate, 0.1 M β-mercaptoethanol, and 1 mg/ml spermidine DNA). After chloroform extraction and ethanol precipitation, DNA was cleaved by the addition of 10%, vol/vol piperidine (Sinopharm Chemical Reagent Co.,Ltd, China) and incubated at 90°C for 30 min. DNA was purified again by chloroform extraction and ethanol precipitation and dissolved in 80% (vol/vol) deionized formamide (Sangon Biotech Co., Ltd, China). The DNA samples were boiled at 95°C for 5 min and subsequently cooled on ice for 15 min before being loaded onto a 20% denaturing polyacrylamide gel. DNA fragments were visualized by iBright 1500 (Thermo Fisher Scientific, USA) and digitized by ImageQuant 5.2 software.

Treated cells were lysed using RIPA buffer as described previously [30 (link)]. Protein samples were loaded into SDS-PAGE gels and transferred to nitrocellulose membrane. The primary antibodies were applied overnight at 4 °C at 1:1000 dilutions after blocking with 5% skim milk solution. The secondary antibodies were applied at 1:5000 for 2 h at room temperature. The bands were developed using ECL reagent (Advensta, Menlo Park, CA, USA), images were captured with a chemi-doc image analyzer (iBright 1500, Thermo Fisher Scientific), and the values were quantified with Image J software (Wayne Rasband, retired from NIH).