Trans blot turbo transfer pack membranes

Kidneys were homogenized in lysis buffer (9803; Cell Signaling Technology, Danvers, MA) containing 1.0 mmol/l phenylmethylsulfonyl fluoride (Thermo Fisher Scientific) and protease inhibitor cocktail (Roche, Basel, Switzerland). Twenty microgram of proteins mixed with Laemmli sample buffer (Bio-Rad) and 2.5% mercaptoethanol were separated by 4–20% Mini-PROTEAN TGX Gels (Bio-Rad) and transferred onto trans-blot turbo transfer pack membranes (Bio-Rad). After incubation with a blocking reagent (PVDF Blocking Reagent for Can Get Signal; Toyobo), the membranes were incubated with antigen-specific antibodies (Table S2, Supplemental Digital Content) overnight at 4°C. Immunoreactive signals were visualized using a horseradish peroxidase-conjugated secondary antibody (1:5000; Santa-Cruz Biotechnology, Dallas, Texas, USA), an enhanced chemiluminescence system (Thermo Fisher Scientific), and Amersham Imager 600 (GE Healthcare, Buckinghamshire, UK). The relative expression level of each protein was normalized to GAPDH.

The cells were washed with PBS and scraped using a cell scraper. The cells were transferred to 1.5 mL microtubes and centrifuged at 5000 g for 3 min. Cells were lysed in 100 μL of lysis buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 6% 2‐mercaptoethanol, 10% glycerol, 0.0125% bromothymol blue) and heated at 95°C for 5 min. Whole cell extracts were subjected to electrophoresis on 4%–20% Mini PROTEAN TGX gels (Bio‐Rad, 4561095) and then transferred to Trans‐Blot Turbo Transfer Pack membranes (0.2 μM PVDF, BIO‐RAD, 1704156). Membranes were blocked with 5% BSA in Tris‐Buffered Saline‐Tween, TBST 0.001% azide for 60 min and probed with primary anti‐STING (D2P2F) rabbit mAb (#13647, Cell Signaling Technology), anti‐cGAS (D1D3G) rabbit mAb (#15102, Cell Signaling Technology), anti‐EGF‐Receptor (D38B1) XP rabbit mAb (#4267), and anti‐β‐actin (13E5) rabbit mAb (#4970, Cell Signaling Technology). The membranes were then washed with TBST and incubated with a secondary donkey anti‐rabbit immunoglobulin antibody conjugated to horseradish peroxidase (HRP) (Abcam, ab205722) for 1 h. Proteins were detected using a chemiluminescence detection system (ECL Prime Western Blot Detection System; GE Healthcare, RPN2232) according to the manufacturer's instructions.