Ab129068

LV protein was extracted by homogenization with ice‐cold RIPA buffer, as previously described (Zhao et al.,2010), and 20 μg loaded onto a 10% SDS‐PAGE gel before blotting on a PVDF membrane (Immobilon‐FL; Millipore). Membranes were incubated overnight at 4°C with a rabbit monoclonal antibody against Nox2 (1:1000, Abcam ab129068) using hypoxanthine phosphoribosyltransferase (HPRT) antibody (1:10 000, ab109021 Abcam) as a loading control. This was followed by incubation with horseradish peroxidase‐labelled goat anti‐rabbit secondary antibody (1:10 000 Cell Signalling Technology #7074P2) for 60 min at room temperature, before the membrane was developed in a darkroom using Immobilon Western Chemiluminescent HRP Substrate (Millipore), scanned and quantified by densitometry (ImageJ). Variations in band density are expressed as fold changes compared with the HPRT control.

For western blotting, cell extracts were prepared by addition of ice cold RIPA buffer (300 μL per well of a 12‐well plate; 1 mL per T25 or T75 cell culture flask) containing protease inhibitor cocktail (200 μL·40 mL⁻¹). Cells were scraped from the adherent layer, the cell suspension was centrifuged at 12000 g for 15–20 min at 4°C and the supernatant analysed for protein before western blot analysis was performed as described above using primary antibodies detecting Nox2 (1:1000, Abcam ab129068) and Mfn2 (1:1000 Abcam ab50838). For measurement of ROS production, homogenates were prepared by probe sonication of the whole cell preparation on ice for 20 s and lucigenin‐enhanced chemiluminescence determined as above.

Western blotting was used to measure the levels of protein expression of AT1R (ab124734), IMD (ab198273), RAMP1/2/3 ((ab-156575, ab-198276, ab-197372), NOX2 (ab129068) and NOX4 (ab133303), antibodies from Abcam, Burlingame, CA, USA) and ERK (T-ERK (16443-1-AP) antibody from Proteintech Group, Inc. Rosemont, IL, USA; P-ERK (AP1015) and CRLR (DF10203) antibodies from Affinity Biosciences. Cincinnati, OH, USA) activation in the PVN. The methods were as described previously [11 (link),19 (link)]. Protein loading was controlled by probing all blots with GAPDH antibody (antibody from Bioworld Technology, Louis Park, MN, USA) and the protein intensity was normalized to that of GAPDH.

Tissues or cardiomyocytes were sonicated in RIPA lysis buffer containing 1% NonidetP-40, 0.1% SDS, protease inhibitor, while adding phosphatase inhibitors cocktail (Bimake, Houston, USA), and then homogenized. The nuclear and cytosolic protein were extracted by commercially available kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells expanded sufficiently under low osmotic pressure, then the cell membrane ruptured, releasing cytoplasmic proteins. Nucleus precipitation could be centrifuged to obtain after then. Finally, the nuclear protein was extracted by high salt nucleoprotein extraction reagent. Equal quantities of tissues or cardiomyocytes protein lysates were subjected to SDS-PAGE (Bio-Rad), and transferred onto PVDF membrane. Blocking was made at room temperature with 5% nonfat milk powder prepared in Tris-buffered saline containing 0.1% Tween 20. Then, membranes were incubated overnight at 4 °C with corresponding antibodies, and followed by incubation with appropriate secondary HRP-conjugated antibodies. Antibodies against p38 (#8690), P-p38 (#4511), NFκB-p65 (#8242), ERK (#4695), P-ERK (#4370), P-Stat3 (#9145), Stat3 (#4904) and GAPDH (#2118) were obtained from Cell signaling Technology (Danvers, USA). Antibodies against FNDC5 (ab174833), P-JAK2 (ab195055), JAK2 (ab108596), Nox2 (ab129068), Nox4 (ab154244) were obtained from Abcam (Cambridge, UK).

The following reagents were used in the study: picroside II (CAS No. 39012-20-9, C₂₃H₂₈O₁₃, 512.48, Tianjin Kuiqing Med. Tech. Co. Ltd.); apocynin (CAS No. 498-02-2, C₉H₁₀O₃, 166.17, Sigma Aldrich); trans-4-bromine cinnamon acid (TBCA, CAS No. 1200-07-3, C₉H₇BrO₂, 227.05, Sigma Aldrich); 2,3,5-triphenyl tetrazolium chloride (TTC, Chinese Chemical Reagent Co., Ltd.); phenylmethylsulfonyl fluoride (PMSF, no. 329-98-6, Beijing Solarbio Tech. Co. Ltd.); an enhanced BCA protein assay kit (No. P0010, Beyotime Institute of Biotech., China); mouse anti-Rac1 monoclonal antibody (Abcam, ab33186), rabbit anti-Nox2/gp91phox monoclonal antibody (Abcam, ab129068), rabbit anti-ROCK1 monoclonal antibody (Abcam, ab45171), anti-MMP2 (Abcam, ab92536), anti-MLCK (Abcam, ab76092), anti-claudin-5 (Santa Cruz Biotech., Lot# L2013); goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (AB136817, Abcam, USA); rabbit anti-β-actin antibody (BA2305, Wuhan Boster Biological Co., Ltd.); rat NADPH oxidase ELISA kit (RG3022) and rat ROS ELISA kit (RG3054) form Trust Specialty Zeal; and Evans Blue (EB, CAS:314-13-16, Sigma Aldrich).

The expression of BMAL1 protein and proteins related to oxidative stress and autophagy was determined by the WB technique as previously described [17 (link)]. Total proteins were extracted by using RIPA lysis buffer supplemented by a protease inhibitor cocktail (Complets, Roche). The equivalent (50 μg) protein was transferred onto the PVDF membrane (IPFL00010; Millipor; USA) after 10% SDS-PAGE electrophoresis. Then, the membranes were blocked with 5% skimmed milk at room temperature and incubated overnight at 4 °C with primary antibodies. The antibody reagents were BMAL1 (ab3350; Abcam; USA,), β-Actin (AP0060; Abcam), Gp91phox (ab129068, Abcam) p67phox (3923, Cell Signaling technology), SOD2 (ab38155, Abcam), HO-1 (ab13243, Abcam), LC3 (12741, CST, USA), P62 (23214, CST), ATG 5,12 (AAM79-1, AbD Serotec, UK), ATG 7(2631, Cell Signaling technology) and Beclin1 (3738, Cell Signaling technology). HRP-conjugated secondary antibodies were then applied to bind and visualize the primary antibodies. Finally, images were obtained by Odyssey Infrared Imaging System (LI-COR Biosciences, USA) to quantify protein expression.