Anti p47phox

Stored artery segments were homogenized in lysis buffer containing 50 mM glycerophosphate, 100 µM sodium orthovanadate, 2 mM magnesium chloride, 1 mM EGTA, 0.5% Triton X-100, 1 mM DL-dithiothreitol, 20 µM pepstatin, 20 µM leupeptin, 0.1 U/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride and then incubated on ice for 1 hr. The sample was centrifuged at 1,000 g for 15 min at 1°C, and the supernatant was collected. The total protein was measured by a BCA kit (Bio-Rad). To examine each protein expression, equal amounts of protein (50 µg) from each sample were loaded on each lane and electrophoresed in 4–20% Tris-Glycine gel (Invitrogen) and then transferred onto a polyvinylidene difluoride membrane (Millipore). After being incubated for 2 hrs in 6% dried milk in TBS-Tween buffer, the membrane was incubated overnight at 4°C with specific primary antibody in blocking buffer (anti-p47^phox and anti-gp91^phox, Santa Cruz Biotech at 1∶200 dilution, MnSOD 1∶1000 dilution, Enzo; eNOS 1∶300 dilution, BD). The membrane was then rinsed and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 hrs at 1∶5,000 dilutions in blocking buffer. All samples from each group were also probed with anti-GAPDH antibody (1∶5000, Abcam) to correct for sample loading.

High-purity grade (≥98%) caffeine and crystal violet were purchased from Aladdin Bio-Chem Technology, Co., Ltd. (Shanghai, China). NADP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and a NADP/NADPH Quantification Kit were obtained from Sigma-Aldrich (St. Louis, MO, United States). D-Glucose 6-phosphate, recombinant G6PDH protein, and the G6PDH Activity Colorimetric Assay Kit were purchased from BioVision Incorporated (Milpitas, CA, United States). Reactive Oxygen Species Detection Reagents and DMEM were purchased from Thermo Fisher Biochemical Products (Beijing) Co., Ltd. FBS and a mixed P/S were purchased from Biological Industries (Kibbutz Beit Haemek, Israel) and Solarbio (Beijing, China), respectively. Primary antibodies against G6PDH, NOX4, NOX2, SOD2, and Cyclin D1 were obtained from Abcam (Cambridge, MA, United States). Antibodies against STAT3, p-STAT3, Ki67, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, United States). Anti-Catalase, anti-p47-phox, and anti-Cyclin E antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Anti-β-tubulin and horseradish peroxidase-conjugated secondary antibodies were purchased from Sino Biological, Inc. (Beijing, China) and R&D Systems (Minneapolis, MN, United States), respectively.

Reagents for SDS-PAGE were obtained from Bio-Rad (Richmond, CA, USA); dihydroethidium (DHE), RPMI 1640 culture medium, heat-inactivated fetal bovine serum, Opti-MEM medium, Lipofectamine 2000, RIPA lysis buffer and Fura-2 AM were obtained from Thermo Fischer Scientific (Waltham, MA, USA); Bradford Reagent and linoleic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA); Insulin Ultra-Sensitive Assay kit was obtained from Cisbio (Codolet, France); GW9508 was obtained from Tocris Bioscience (Bristol, England, UK); VAS2870 was obtained from Enzo Life Sciences (Farmingdale, NY, USA); p22^phox siRNA (#sc-61892), control siRNA (#sc-37007), anti-p47^phox (#sc-14015; dilution 1:1000 in 5% BSA in TBST), anti-p22^phox (#sc-271968; dilution 1:500 in 5% BSA in TBST) and anti-CD73 (#sc-25603; dilution 1:1000 in 5% BSA in TBST) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); goat anti-rabbit IgG HRP (#ab205718; dilution 1:10,000 in 3% skimmed milk in TBST) and goat anti-mouse IgG HRP (#ab97023; dilution 1:10,000 in 3% skimmed milk in TBST) secondary antibodies were obtained from Abcam (Cambridge, UK).

Cytosol to membrane translocation of p47phox, which is essential for NADPH oxidase activation, and Rac1/2 (small G protein necessary for NADPH oxidase activity) was assessed in VSMCs. Cells were lysed and fractionated to obtain cytosol- and membrane-enriched fractions. Western blotting was performed as described using anti-p47phox (Santa Cruz Biotechnology, TX-USA) and anti-Rac1/2 (Cell Signaling, MA-USA). Translocation was determined as the ratio of protein expression in membrane to cytosolic fractions.

The heart and liver tissues were fixed in 10% (w/v) neutral buffered formalin and embedded in paraffin, and 4 μm-thick sections were prepared. The sections were deparaffinized with xylene, and endogenous peroxidase activity was blocked with hydrogen peroxide. Next, the cells were washed with phosphate buffer and treated overnight with anti-CD68 (Abcam plc, Cambridge, UK) or anti-p47phox (Santa Cruz Biotechnology, Inc., Dallas, CA, USA) antibodies. They were then treated with the secondary antibody MAX-PO (MULTI) (Nichirei Biosciences Inc., Tokyo, Japan). Color was developed using the peroxidase substrate diaminobenzidine (ImmPACT TM DAB; Vector Laboratories, Inc., Burlingame, CA, USA) [52 (link)]. Immunostaining was quantified by randomly imaging CD68 (ten fields of liver tissue at 100×) or p47phox (20 fields of liver tissue at 400×) staining using an optical microscope equipped with a high-resolution video camera (CX41; Olympus, Tokyo, Japan). Next, the ratio (%) of the positively stained region (brown area) to the total area of the liver tissue was calculated. All images were analyzed using the ImageJ software (version 1.52, National Institutes of Health, New York, NY, USA).

Cells from cultures were washed three times in PBS and incubated in lysis buffer (50 mM Tris–Hcl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.02% sodium azide, 100 μg/ml PMSF, 1 μg/ml peptin, and 1 μg/ml aprotinin) for 30 min on ice. The homogenate was centrifuged at 16,000 g for 45 min at 4°C. An equal amount of protein from each lysate was subjected to 12% SDS–PAGE and transferred onto a nitrocellulose membrane. After blocking for 1.5 h in 5% nonfat dried milk containing 0.1% Tween-20, the membrane was incubated at 4°C overnight with the following antibodies: rabbit polyclonal anti-p47phox (1:100, Santa Cruz, CA, USA). The membrane was washed and further incubated for 1 h at room temperature with HRP-conjugated secondary Abs. Following washing, immunoreactive bands were detected using chemiluminescence reagent (Amersham Biosciences).