P22phox

Proteins were extracted from each group of left ventricular tissues with lysis buffer supplemented with protease inhibitor and phenylmethylsulfonyl fluoride (PMSF). Proteins (20 μg /sample) were separated by electrophoresis with 10% SDS/PAGE gels. After electrophoresis, proteins were transferred to nitrocellulose membranes. The blots were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with primary antibodies against mouse LOX-1 (a gift from Dr. Sawamura; 1:2,000, v/v, dilution), fibronectin (Abcam; 1:2,000, v/v, dilution), collagen-1a (Santa Cruz, Dallas, TX, USA; 1:1,000, v/v, dilution), collagen-3a (Santa Cruz; 1:1,000, v/v, dilution), collagen-4a (Santa Cruz; 1:1,000, v/v, dilution), p22^phox (Santa Cruz; 1:1,000, v/v, dilution), gp91^phox (Abcam; 1:2,000, v/v, dilution) or β-actin (Santa Cruz; 1:2,000, v/v, dilution) in TBS-T at 4°C overnight on a shaker. After washing with TBS-T three times, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) for 1 h at room temperature. The blots were scanned with a ChemiDOC XRS system (Bio-Rad, Hercules, CA, USA) following exposure to Luminol Reagents (Beyotime, Shanghai, China) for 3 min.

The following antibodies were used for immunoblotting analysis using standard Western blotting procedures: superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), p22phox, and p-p40phox were purchased from Santa Cruz Biotechnology, Dallas, TX, USA; β-actin, tublin, diphenyleneiodonium chloride (DPI), and capsaicin were purchased from Sigma-Aldrich, St. Louis, MS, USA.

The preparation of protein samples from myocardial tissues was performed as previously described (Becker et al. 2005 (link)). Antibodies to eNOS (BD Transduction Laboratories, 1:250 dilution), phospho-eNOS (Ser 1177) (Cell Signal, 1:500 dilution), SOD-1(Calbiochem, 1:5000 dilution), SOD-2 (BD Transduction Laboratories, 1:10,000 dilution), SOD-3 (Santa Cruz Biotechnology, 1:5000 dilution), or one of the following subunits of NAD(P)H oxidase: p67^phox (Upstate, 1:1000 dilution), p22^phox (Santa Cruz Biotechnology, 1:2000 dilution), gp91^phox(BD Transduction Laboratories, 1:1000 dilution), p47^phox (Santa Cruz Biotechnology, 1:1000 dilution), phos-p47^phox (Upstate, 1:1000 dilution), p40^phox (Santa Cruz Biotechnology, 1:800 dilution), Rac-1 (Santa Cruz Biotechnology, 1:5000 dilution), and nitrotyrosine (Santa Cruz Biotechnology, 1:1000 dilution) were used.

Binding of CDDP to p22phox recombinant proteins was validated by GST pull-down-based binding assay. Briefly, GST, GST-p22phox FL or GST-p22phox CT protein (16 μg each) was immobilized to the GSH sepharose beads at 4 °C for 1 h. The protein-bound beads were incubated in the PBST buffer with or without Alexa Fluor 488-labeled CDDP (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 1 h. After washing the beads with the PBST buffer, the CDDP-bound protein samples were eluted by the SDS sample buffer. The samples were then analyzed by dot blot or Western blot analysis using antibodies against p22phox (Santa Cruz, CA, USA) or Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA). For cell-based binding assay, after treated with Alexa Fluor 488-labeled CDDP overnight, the p22phox-expressing SAS cells were lysed in RIPA buffer and cell lysates were collected. The lysates (200 μg) were then incubated with Protein A/G beaded agarose and anti-p22phox antibody (10 μg) at 4 °C overnight. After washing with RIPA buffer, the immunoprecipitates were analyzed by immunoblotting using anti-Alexa Fluor 488 antibody.

Cells were lysed in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, 5 mM NaF and 5 mM Na₃VO₄. Total cell lysates (30 µg) were resolved by 10% SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted using 100–200 µg/mL antibodies, including p-ERK/t-ERK, NF-κB p-p65/t-p65, Rac1 (cell signaling), Nox1, HSPG (Abcam), p22^phox, p47^phox, Nox2, Nox4 (Santa Cruz). For loading controls, blots were reacted with antibodies detecting β-Actin (Sigma). Immunoreactive bands were developed using an enhanced chemiluminescence reaction (Perkin-Elmer) and visualized by autoradiography.

Cells were lysed in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, 5 mM NaF and 5 mM Na₃VO₄. Total cell lysates (30 μg) were resolved by 10% SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted using 100–200 μg/mL antibodies, including cleaved-caspase 3/9, p-Akt/t-Akt, p-GSKα/t-GSKα, p-P70S6/t-P70S6, p-S6/t-S6, XIAP, Rac1 (cell signaling), Nox1 (Abcam), xCT, p22^phox, p47^phox, Nox2, Nox4 (Santa Cruz), KSHV-K8.1 (ABI). For loading controls, blots were reacted with antibodies detecting β-Actin (Sigma). Immunoreactive bands were developed using an enhanced chemiluminescence reaction (Perkin-Elmer) and visualized by autoradiography.