Ecltm prime western blotting detection reagent

Recombinant proteins (15 μg/well) were separated in 8% Tricine SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes at 30 V overnight. Membranes were blocked with 5% of non-fat dry milk for 2 h and incubated overnight with rabbit hyperimmune sera (1:1000). The next day, membranes were washed with PBST and incubated for 4 h with a goat anti-rabbit IgG peroxidase-coupled secondary antibody (1:40,000) (Sigma). All the membranes were revealed using ECL^TM Prime Western Blotting Detection Reagent (GE Healthcare) following the manufacturer’s recommendations.

To extract protein, ahRPE were washed and then lysed with RIPA buffer containing phosphatase and protease inhibitor cocktails (Roche) on ice. Samples were scraped on ice, agitated for 30 min at 4 °C and then centrifuged at 12,000 rpm for 20 min at 4 °C to remove debris. The supernatant was collected, aliquoted to prevent freeze-thaw cycles, and protein concentration was subsequently determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts were separated by Bolt^TM 4–12% Bis-Tris Plus Gels (Thermo Fisher Scientific) and transferred onto iBlot® 2 PVDF Mini Stacks (Thermo Fisher Scientific) membranes and were probed with antibodies (Supplementary Table 4). Proteins of interest were detected with Peroxidase AffiniPure Goat Anti-Mouse IgG, F(ab’)₂ Fragment Specific antibody (1:10000, Jackson ImmunoResearch, Cat #:115–035–072) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) antibody (1:10000, Jackson ImmunoResearch, Cat #:111–035–045), and subsequently visualized with ECL^TM Prime Western Blotting Detection Reagent (GE Healthcare) according to the provided protocol. All samples were stored at −80 °C. Uncropped western blots for Figs. 3d, 5c, d, and 6d can be found in Supplementary Figs 5 and 6.

Whole-cell lysate samples were separated by SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore, Bedford, MA, USA) by electroblotting at 15 V for 60 min. For blocking, the membrane was incubated in Tris-buffered saline containing 0.05% Tween 20 and 3% BSA (TBST-3% BSA). Blots were probed with appropriate antibodies (Supplementary Table 1), and the signals were visualized by chemiluminescence. All antibodies were used at a 1:1000 (first antibodies—Rabbit polyclonal anti-EGFP; Cat# A11122; Life Technologies: Rabbit polyclonal anti-α-tubulin; Cat# ab15246; Abcam, Cambridge, MA, USA) or 1:2,000 (second antibody—Donkey anti-rabbit IgG-horseradish peroxidase (HRP)-conjugate; Cat# NA934V; GE Healthcare, Buckinghamshire, UK) dilution in TBST-0.1% BSA for 1 h at room temperature. After washing by TBST for 1 h at room temperature, HRP-dependent luminescence was developed with ECL^TM Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a multi-imaging Analyzer Fusion Solo 4^TM system (Vilber Lourmat, Eberhardzell, Germany). Uncropped scans of the blots were shown in Supplementary Fig. 3.

Whole-cell lysates were prepared from cells treated with 1.5 μM CX-5461 for 24 h or medium as control, by addition of cell lysis buffer (#9803, Cell signaling, Leiden, The Netherlands) diluted in demineralized water and supplemented with sodium orthovanadate (S6508, Sigma-Aldrich, Zwijndrecht, The Netherlands) and protease inhibitor cocktail (11697498001, Sigma-Alrich, Zwijndrecht, The Netherlands), followed by incubation on ice for 30 min. The samples were spun down at 16,000× g for 10 min at 4 °C and the supernatant was used to perform the Bio-rad protein estimation using the protein assay dye reagent concentrate (#500-0006, Bio-Rad, Veenendaal, The Netherlands). Finally, 50 μg protein samples were separated on Mini-Protean TGX^TM precast gels and transferred to PDVF membranes prior to target detection on the Uvitec using ECL^TM Prime Western blotting detection reagent (lot#13601176, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
Antibodies: Primary, Phospho-Histone H2A.X (#2577, Cell signaling, 1:1000), β-actin (#4967S, Cell signaling, 1:2000). Secondary, Anti-rabbit IgG HRP linked (#7074, Cell signaling, 1:2000), Anti-mouse IgG HRP linked (#7076, Cell signaling, 1:2000).

Cells were seeded in 6-well plates (seeding density 1 × 10⁶ cells/well) and incubated with compounds for 48 h. Cell lysates were prepared by the RIPA buffer (Sigma-Aldrich, Germany) and the total protein concentrations were determined by the Bradford assay prior to western blot. The signal proteins on the membrane were detected using ECLTM Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, United Kingdom) representing a horseradish peroxidase substrate for enhanced chemiluminescence (ECL) and signal detection. Protein bands were visualized by Molecular Imager ChemiDocTM XRS + Imaging System (Bio-Rad, United States) and normalized using the Image Lab software (v.6.0). Antibodies were purchased from Cell Signaling Technology, Netherlands. Primary antibodies used were PARP Antibody #9542, Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit mAb #2060, Phospho-p70 S6 Kinase (Thr421/Ser424) Antibody #9204, Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033, Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb #4060, PTEN (D4.3) XP^® Rabbit mAb #9188, Mcl-1 Antibody #4572, and Cyclin D1 Antibody #2922. For loading control, β-Actin (8H10D10) Mouse mAb #3700 primary antibody was used. Secondary antibodies anti-rabbit IgG, HRP-linked Antibody #7074, and anti-mouse IgG and HRP-linked Antibody #7076 were used.

The expression of ABCC11 protein in plasma membrane vesicles was examined by immunoblotting, as described previously [4 (link),9 (link)] with minor modifications. Briefly, the prepared samples were electrophoretically separated on poly-acrylamide gels and transferred to a Hybond^® ECL^TM nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min. After blocking by Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (TBST-skim milk) at 4 °C overnight, blots on the membrane were probed with a rat monoclonal anti-ABCC11 antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal anti-Na⁺/K⁺-ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1000 fold), followed by incubation with a goat anti-rat immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugated antibody (NA935V; GE Healthcare; diluted 2000 fold) and a donkey anti-rabbit IgG–HRP conjugated antibody (NA934V; GE Healthcare; diluted 3000 fold), respectively. All antibodies were used in TBST-skim milk. HRP-dependent luminescence was developed using the ECL^TM Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a multi-imaging Analyzer Fusion Solo 4^TM system (Vilber Lourmat, Eberhardzell, Germany).

Retinas were fresh dissected and immediately frozen in dry ice. Then, retinas were homogenized in lysis buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 2% Triton X-100) supplemented with 100 μl/ml of protease inhibitor mixture (Sigma-Aldrich, Madrid, Spain) for 2 min at 50 Hz using the mechanical homogenizer TissueLyser LT (Qiagen). Lysates were incubated 30 min on ice and centrifuged to remove particulate matter. Protein concentration was determined with the Bradford assay (Bio-Rad, Hercules, CA). A total of 80 μg of protein were resolved in 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) by electroblotting. Blots were blocked for 1 h with 5% skim milk in PBS containing 0.5% Tween-20 (PBS-T, pH 7.4) and then were incubated overnight at 4 °C with anti-P2X7 receptor C-terminal rabbit affinity isolated polyclonal antibody (Alomone Labs) at 1:1000 dilution followed by incubation with HRP-conjugated secondary anti-rabbit (GE Healthcare) at 1:5000 dilution. As loading control β-actin was identified using anti-β-Actin-HRP mouse monoclonal antibody (clone C4, Santa Cruz Biotechnology, Heidelberg, Germany). Membranes were developed using ECL^TM Prime Western Blotting Detection Reagent (GE Healthcare) and the density of the bands of proteins were quantified using image analyzer Chemidoc^TM XRS + (Bio-Rad, Hercules, CA) and the software (ImageLab^TM 5.2.1).