Image lab system

Protein expression of mitochondrial respiratory Complexes I-V was measured by Western immunoblot. Mitochondrial protein fractions obtained from left ventricular heart tissues and cardiomyocytes from separate groups of prenatally NMX and HPX 90 d old offspring were run on separate gels. Mitochondrial proteins (4 μg) were separated on 4-15% precast gradient gels (Bio-Rad) and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in TBST for 2 hrs, incubated overnight at 4°C with primary antibody diluted in 5% nonfat milk in TBST, and then detected using an appropriate peroxidase-conjugated secondary antibody. Protein bands were targeted with an antibody cocktail (1 : 500) containing antibodies for complex subunits (I: NDUFB8, 20 kDa MW; II: SDHB, 30 kDa MW; III: UQCRC2 48 kDa MW; IV: MITCO1, 40 kDa MW; and V: ATP5a, 55 kDa MW) (Abcam, Cambridge, MA) and polyclonal anti-VDAC (voltage-dependent anion channel) antibody (1 : 2,000, Boster Biological Technology Co., Pleasanton, CA) and visualized by the ChemiDoc Touch Imaging System (Bio-Rad). Band densities were quantified by the Bio-Rad Image Lab System and normalized to the loading control, VDAC, to confirm equal loading.

For protein extraction, HepIR cells were homogenized in 100 µL RIPA buffer (P0013B, Beyotime). Proteins were transferred to polyvinylidene difluoride membrane and incubated with a blocking buffer (5% BSA in 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Membranes were incubated with primary antibodies for 16 h at 4°C, washed three times for 10 min each and then incubated with second antibodies for 1 h at room temperature. Signals were detected using the ChemiDoc™ XRS+ and the Image Lab™ system (BIO-RAD, United States).

Cells or tissues were lysed in RIPA lysis buffer (P0013B, Beyotime) supplemented with protease inhibitor (4693159001, Roche) and phosphatase inhibitors (4906837001, Roche). Protein concentration was measured using a BCA protein assay kit (23225, Thermo Fisher Scientific). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system (Bio-Rad). Membranes were incubated for 1 hour at room temperature in blocking buffer (5% BSA, FA016, GENVIEW), then incubated at 4°C overnight with primary antibodies. Antibodies were incubated with horseradish peroxidase–conjugated secondary antibodies. The primary antibodies are described below. The secondary antibodies were from Promega (W4011, W4021). Signals were detected using ChemiDOC XRS+ and the Image Lab system (Bio-Rad).

Total protein was isolated from FDB by homogenization in lysis buffer (20 mM HEPES buffer, pH 7.5, 100 mM NaCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 20% glycerol) containing 1 mM DTT and a protease inhibitor cocktail (Sigma) on ice. Samples were centrifuged at 20,000 g at 4 °C, and the supernatant was collected and frozen at − 80 °C until analyzed. Total protein concentration was determined using a BCA assay (Thermo Fisher Scientific). Samples were solubilized in SDS loading buffer and denatured by heating at 100 °C for 5 min. For immunoblotting, 20–30 μg total protein was loaded on bis-acrylamide gels, separated by SDS-PAGE electrophoresis, and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk at room temperature for 1 h followed by overnight incubation at 4 °C in anti-SERCA1 (Thermo Fisher Scientific) or anti-calsequestrin polyclonal antibodies (Abcam ab3516). Antibodies against α-tubulin or actin (Abcam) were used as loading controls. Antibody-reactive proteins were detected with Clarity western ECL substrate (Bio-Rad, Hercules, CA, USA), imaged using an Image Lab system (Bio-Rad), and quantified by densitometry.

Mouse tail tissue was collected, and genomic DNA was isolated, mixed and incubated with a commercial kit DNeasy® Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. A final centrifugation for 1 min at 6000×g was performed and the concentration of total DNA was measured by a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). The primers are listed as below.
NLRP12 primers:
Forward: TGGCTTCTATTCAACTCCCT.
Reverse: ATCGTTACACTCGGCTTCTC.
GSDMD primers:
Forward: CGATGGAACGTAGTGCTGTG.
Reverse: TCCTTCCCAACCTGCTGTTG.
The DNA sequences were verified by PCR and sequencing.
After PCR amplification, the samples were electrophoresed in 2% agarose 1 × TAE (Tris [40 mM Tris], acetic acid [20 mM] and ethylenediaminetetraacetic acid [1 mM]) gels, and stained with 1 × SYBR® Safe DNA Gel Stain (Life Technologies). Images were collected by an Image Lab system (Bio-Rad).