P67phox

Membrane proteins or total cell proteins were extracted from homogenized LV tissue. The primary antibodies included NOX2, NOX4, p22^phox, p47^phox, p67^phox, Rac-1 (1 : 500, Abcam); eNOS, p-eNOS^Ser1177, p-eNOS^Thr495, p-eNOS^Ser114, iNOS, nNOS (1 : 1000, Cell Signaling Technology), β3-adrenergic receptor (1 : 500, Abcam), and β-actin (1 : 5000, Abcam).

Proteins from sciatic DRG were extracted using RIPA buffer with protease and phosphatase inhibitor cocktails (Roche). Total lysates were obtained by 30 min lysis on ice followed by 30 min centrifugation at 4 °C. Protein concentration of lysate was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Ten to 50 μg proteins were loaded to SDS-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes for 2 h. Membranes were blocked with 5% bovine serum albumin (BSA) or milk for 1 h at RT and incubated with gp91phox (1:200, Mouse, BD Biosciences), p47phox (1:200, Rabbit, Santa Cruz), p-p47phox (S345) (1:100, Rabbit, Sigma), p67phox (1:500, Rabbit, Abcam), p22phox (1:100, Rabbit, Abcam), H3 (1:1000, Rabbit, Abcam), STAT3 (1:500, Mouse, Cell Signaling Technology), pSTAT3 (1:1000, Rabbit, Cell Signaling Technology), pPKC (1:1000, Rabbit, Cell Signaling Technology), PKC (1:1000, Rabbit, Abcam), or GAPDH (1:1000, Rabbit, Cell Signaling Technology) at 4 °C O/N. Following incubation with horseradish peroxidase-linked secondary antibody (1:1000 anti-Rabbit or anti-Mouse GE Healthcare) for 1 h at RT, membranes were developed with ECL substrate (Thermo Scientific).

MHT granules, consisted of HE granule (39.4%), RC granule (26.3%), SAA granule (15.5%) and RG granule (18.8%), were provided by Guangdong Yi Fang Pharmaceutical Co Ltd (Guangzhou, China). Rhodamine 6G was from Fluka Chemie AG (Buchs, Switzerland). Antibodies against Cav-1, phosphor-Cav-1, Src, phosphor-Src, p-VE-cadherin, β-actin, β-tubulin and Histone-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against JAM-1, Claudin-5, ZO-1, NF-κB p65, Na+/K+-ATPase, NADPH oxidase subunit p47phox and p40phox, VE-cadherin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Occluding, NADPH oxidase subunit p67phox were purchased from Abcam (Cambridge, UK).

40 μg of protein from heart tissue or cell lysates or 10 μg of protein from isolated membrane lysates were loaded onto SDS-polyacrylamide gels. After electrophoresis, the separate proteins were transferred onto Bio-Rad polyvinylidene difluoride membranes. After blocking in 5% nonfat milk for 1 h, the membranes were incubated with antibodies against NOX4 (1:1000 dilution; Abcam), Rac1 (1:1000 dilution; Abcam), MuRF1 and atrogin-1 (1:1000 dilution; Abcam), Na⁺/K⁺-ATPase (1:1000 dilution; Abcam), p67^phox (1:1000 dilution; Abcam), phosphorylation of Ser-345 and Ser-370 on p47^phox and total p47^phox (1:1000 dilution; Thermo Fisher Scientific Inc.), phosphorylated p38 and total p38 (1:1000 dilution; Cell Signaling Technology), phosphorylated ERK1/2 and total ERK1/2 (1:1000 dilution; Cell Signaling Technology), and GAPDH (1:5000 dilution; Cell Signaling Technology), respectively, followed by secondary relevant antibodies conjugated with horseradish peroxidase. The signals were then developed using an enhanced version of the chemiluminescence reaction.
The protein ladders were purchased from FroggaBio Inc. (Concord, Canada) for cultured cardiomyocytes (245, 180, 135, 100, 75, 63, 48, 35, 25, 20, 17, and 11 kDa) and Thermo Fisher Scientific China Co. Ltd. for heart tissue samples (170, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kDa).

40 μg of heart tissue lysates or 10 μg of isolated membrane lysates were loaded on sodium dodecyl sulfate polyacrylamide electrophoresis gels. After electrophoresis, the separated proteins were transferred onto Bio-Rad PVDF membranes. The membranes were incubated with antibodies against Rac1 (1:1000 dilution, Abcam), ubiquitin ligase muscle RING-finger protein-1 (MuRF1, 1:1000 dilution, Abcam), Na⁺/K⁺ ATPase (1:1000 dilution, Abcam), p67^phox (1:1000 dilution, Abcam), residual phosphorylation of Ser345 on p47^phox and total p47^phox (1:1000 dilution, Thermo Fisher Scientific Inc.), NOX4 (1:1000 dilution, Abcam) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:5000 dilution, Cell Signaling Technology), respectively. After washing, the membranes were incubated with relevant secondary antibodies conjugated with horseradish peroxidase. The signals were then developed using an enhanced version of the chemiluminescence reaction.

For in vitro acetylation, 1 μg GST-tagged recombinant Rac1 (Cytoskeleton Inc., Denver, United States) was incubated with acetyl-CoA (20 μM, Active Motif) and p300 acetyltransferase (100 ng, Active Motif) at 30°C for 30 min. The sample was then incubated with deacetylation buffer (25 mM Tris•HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂/1 mg/ml BSA), 1 mM NAD⁺, and the active recombinant SIRT1 (100 units; Biozol, MBL Inc., Woburn) at 37°C for 1 h to initiate the deacetylation reaction. The reaction mixture was used for subsequent immunoblotting, pull-down or mass spectrometry analyses. In case of immunoblotting, the reaction mixtures were subjected to SDS-PAGE and immunoblotted with antibodies against SIRT1 (Santa Cruz), Rac1 (Millipore), p67phox (Epitomics), or acetylated lysine (Cell Signaling).