Immobilon western chemiluminescent hrp substrate

The right brain cortex and retina protein samples (10 μg) were subjected to 8–12% SDS-PAGE and then transferred to PVDF membranes (Merk Millipore, Burlington, MA, USA). Blocked with 5% normal goat serum (NGS) for 2 h, the membranes were incubated with primary antibodies: rabbit Anti -NLRP3 (1:1000, Cat# ab263899; Abcam), rabbit anti-GSDMD (1:500, Cat# AF-4012; Affinity), rabbit anti-caspase-1 (1:1000, Cat# AF-4022; Affinity), and rabbit anti-β-actin (1:200; Cat# AF5003; Beyotime Biotechnology, Shanghai, China) at 4 °C for 24 h. The membranes were then incubated with anti-rabbit secondary antibodies (1:500; Cat# A0239; Beyotime Biotechnology, Shanghai, China) at 4 °C for 2 h. The protein signals were detected by Immobilon Western Chemiluminescent HRP substrate (Merk Millipore, Burlington, MA, USA) and analyzed using ImageJ analysis software (https://imagej.net/Fiji/Downloads, accessed on 30 May 2017, Bethesda, MD, USA).

Protein extracts obtained as indicated above were denatured at 95 °C for 10 min, electrophoresed in 7–10% SDS-PAGE and blotted onto nitrocellulose membranes for 3 h at 80 V. The membranes were blocked with either 5% nonfat dry milk or 3% bovine serum albumin (BSA) diluted in TBST (20 mM Tris, 140 mM NaCl, 0.1% Tween, pH 7.6), and incubated overnight at 4 °C, with specific primary antibodies (Supplementary Table S1). Bound antibodies were detected with horseradish peroxidase (HRP)-coupled anti-rabbit or anti-mouse IgG antibodies (Supplementary Table S1) for 1 h at room temperature. After washing in TBST, immunoreactive bands were revealed by the Immobilon^TM Western chemiluminescent HRP substrate (Millipore, Burlington, MA, USA, WBKLS).

Cells were homogenized in radioimmunoprecipitation assay buffer supplemented with Complete Mini protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of protein were resolved by SDS-PAGE, and Western blot were carried out following standard protocols. The primary antibodies used were given in Supplementary Table 7. The HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). Immuno-complexes were visualized with ImmobilonTM Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).

Ventricular tissue from WT, SCN10A^−/−, CaMKIIδc^+/T, and SCN10A^−/−/CaMKIIδc^+/T mice or pellets of iPSC-cardiomyocytes from WT and homozygous Na_V1.8 knockout (KO) lines were homogenized in Tris buffer containing (in mmol/L): 20 Tris-HCl, 200 NaCl, 20 NaF, 1 Na₃VO₄, 1 DTT, 1% Triton X-100 (pH 7.4), and complete protease and phosphatase inhibitor cocktails (Roche Diagnostics). Protein concentration was determined by BCA assay (Pierce Biotechnology). Denatured tissue homogenates (10 min, 70 °C in 2% beta-mercaptoethanol) were separated on 8% SDS-polyacrylamide gels, then transferred to a nitrocellulose membrane, and incubated with the following primary antibodies: rabbit polyclonal anti-CaMKIIδ (1:5000, Thermo Scientific, PA5-22168), rabbit polyclonal anti-Na_V1.5 (1:2000, Alomone labs, ASC-005), and mouse monoclonal anti-GAPDH (1:20000, BIOTREND, BTMC-A473-9) at 4 °C overnight. Secondary antibodies included HRP-conjugated goat anti-rabbit and goat anti-mouse (1:20000, Jackson Immunoresearch, 111-035-144 and 115-035-062, respectively). The membrane was incubated with secondary antibodies for 1 h at RT. ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore) was used for chemiluminescent detection. Analysis was performed using Image Studio Lite Ver 5.2. The full scan blots for Na_V1.5 and CaMKIIδ are shown in the Data Availability file.

The protein extracts were submitted to western blotting standard protocol. Proteins were transferred to poly(vinylidene) difluoride (PVDF) membranes (GE Healthcare). The antibodies used were mouse anti-OCT4A antibody (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-mouse IgG, HRP-linked antibody (Cell Signaling, Danvers, MA, USA) and anti-beta Actin HRP-linked antibody (Abcam). The immunoblots were developed using the ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).