Universal hood 3 imaging system

An equal amount of protein from each group was fractionated by 8%–15% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred onto a PVDF membrane (Millipore, Germany). The membranes were incubated with primary antibody overnight at 4℃, followed by horseradish peroxidase (HRP)‐conjugated secondary antibodies. The antibodies used in this study were anti‐CIRP (Proteintech, 1:500), anti‐caspase‐3 (9662, Cell Signalling Technology, 1:1000), anti‐cleaved caspase‐3 (9661, Cell Signalling Technology, 1:1000), anti‐Fis‐1 (GTX111010, GeneTex, 1:250), anti‐Drp‐1 (ab184247, Abcam, 1:1000), anti‐UCP2 (uncoupling protein 2, 89326, Cell Signalling Technology, 1:1000), anti‐TFAM (22586–1‐AP, Proteintech, 1:200), anti‐TLR‐4 (19811–1‐AP, Proteintech, 1:500), anti‐gp91^phox (19013–1‐AP, Proteintech, 1:1000), anti‐p47^phox (YT3520, Immunoway, 1:1000) and anti‐β‐actin (4967, Cell Signalling Technology, 1:1000). Proteins were detected using Clarity Western ECL substrate (Bio‐Rad Laboratories) and viewed using a Universal Hood III imaging system (Bio‐Rad).

Using 7.5-15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were fractionated and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The PVDF membranes were incubated with the primary antibodies at 4°C overnight, followed by incubation with horseradish peroxidase- (HRP-) conjugated secondary antibodies. Apoptosis was indexed by measuring expression of anti-Bax (MA5-32031, Invitrogen, 1 : 1000), anti-Bcl2 (DF6015, Affinity, 1 : 500), and anti-caspase-3 (ab13847, Abcam, 1 : 500). NADPH oxidase was detected using anti-gp91^phox (19013-1-AP, Proteintech, 1 : 500) and anti-p47^phox (YT3520, Immunoway, 1 : 500). Mitochondrial dynamics was determined by measuring expression of anti-Fis1 (10956-1-AP, Proteintech, 1 : 1000), anti-Drp1 (DF7037, Affinity, 1 : 500), and anti-Mfn2 (12186-1-AP, Proteintech, 1 : 2000). The other antibodies used in this study included TLR-4 (19811-1-AP, Proteintech, 1 : 1000), MyD88 (BA2321, Boster, 1 : 500), and anti-β-actin (4967, Cell Signaling Technology, 1 : 1000). Clarity Western ECL substrate (Bio-Rad Laboratories) and a Universal Hood III imaging system (Bio-Rad) were used to detect the proteins, respectively.