Ab55831

Cell extracts were prepared with a lysis buffer according to the manufacturer's instructions. Forty-microgram proteins were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with NOX-1 (ab55831, Abcam, USA), NOX-2 (ab80508, Abcam, USA), NOX-4 (ab154244, Abcam, USA), RAGE (ab3611, Abcam, USA), or GAPDH (ab8245, Abcam, USA) antibodies at 4°C overnight and subsequently with peroxidase-conjugated secondary antibody for 1 h at room temperature. Proteins were visualised using electrochemiluminescent reagents.

The membrane protein extractions from the aortic media (VSMCs were the only cell type in this layer) or cultured VSMCs were carried out by the Plasma Membrane Protein Isolation Kit (Abcam, UK) according to the manufacturer's instructions. Total proteins were also extracted, and western blot was performed as described in our previous reports [12 (link), 13 (link)]. The expressions of NOX subunits and mitogen-activated protein kinase (MAPK) signaling pathway were detected using their specific antibodies: anti-NOX1 (1 : 2000, ab55831, Abcam, Cambridge, UK), anti-NOX2 (1 : 500, ab80508, Abcam, Cambridge, UK), anti-p47phox (1 : 1000, ab795, Abcam, Cambridge, UK), anti-NADPH oxidase organizer 1 (anti-NOXO1, 1 : 500, sc-390927, Santa Cruz Biotechnology Co., Ltd., Japan), anti-Rac1 (1 : 1000, sc-95, Santa Cruz Biotechnology Co.), anti-p38 (1 : 1000, ab31828, Abcam), anti-phospho-p38 (p-p38, 1 : 1000, ab47363, Abcam), anti-ERK1/2 (1 : 1000, ab17942, Abcam), anti-phospho-ERK1/2 (p-ERK1/2, 1 : 1000, 9101, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-JNK (1 : 1000, ab179461, Abcam), and anti-phospho-JNK (p-JNK, 1 : 1000, ab124956, Abcam). To ensure equal protein loading, β-actin (1 : 5000, ab8226; Abcam) and Na⁺/K⁺-ATPase (1 : 100000, ab76020, Abcam) were used as loading controls for the cytoplasm and plasma membrane, respectively.

Cell were washed with PBS and fixed with 4% paraformaldehyde supplemented with 0.25% Tris-X100 at room temperature for 10 min. After blocking with PBS supplemented with 5% BSA for 2 h at room temperature, cells were incubated with NOX-1 (1:200, ab55831, Abcam), EBP50 (1:1000, 3394, Abcam or 1:200, sc-271552, Santa Cruz Biotechnology), p47phox (1:200, ab166930, Abcam or 1:200, sc-17845, Santa Cruz Biotechnology) at 4° C overnight. Cells were incubated with secondary peroxidase conjugated goat anti-rabbit IgG (1:100, Santa Cruz Biotechnology) antibody for 2 h at room temperature, after washing with PBST for 15 min. Cells were stained with DAPI for 15 min at darkness, after washing with PBST for 15 min. Cell samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Germany).

After conducting the micro-CT analysis, the femurs were preserved at 4°C for histological and immunohistochemical analyses. The right femurs of the 3 groups of mice were first decalcified in 15% ethylenediaminetetraacetic acid (EDTA, Sigma), embedded in paraffin blocks, and sectioned at a thickness of 5 μm using a microtome. Then, the sections were subjected to hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAcP) and immunohistochemical (IHC) staining. The primary antibodies against MMP9 (ab38898, Abcam), NFATc1 (ab25916, Abcam), Heme Oxygenase 1 (HO-1; ab13243, Abcam) and NOX1 (ab55831, Abcam) were used for immunohistochemistry. The stained sections were photographed using a Optical Microscope Olympus CX43 (Tokyo, Japan). The histomorphometric analyses of the bones were performed using the BIOQUANT OSTEO software (Bioquant Image Analysis Corporation, Nashville, TN, USA).

fPAEC were plated at a density of 200,000 cells/well in 6-well plates (Thermo Fisher Scientific). After two days in culture, cells were serum starved for 6 h and treated for 1 or 6 h as mentioned above. Thereafter, cells were collected in 50 μL RIPA lysis and extraction buffer (Sigma Aldrich) containing protease inhibitors (Roche, Basel, Switzerland). Total protein concentration was determined by bicinchoninic acid assay (BCA; Thermo Fisher Scientific). A total of 10 µg of total protein was loaded onto 4–20% SDS-PAGE gradient gels (BioRad Technologies) and resolved at 120 V for 1 h 10 min. Membranes were incubated with antibodies against phospho-p65 NF-κB (3033; Cell signaling, Danvers, MA, USA), total p65 NF-κB (8242; Cell signaling), Hsp90 (610418; BD Bioscience; New Jersey, NJ, USA), Nox1 (ab55831, Abcam, Cambridge, UK) and β-actin (ab6276, Abcam). All antibodies were diluted in 5% non-fat dry milk (BioRad Technologies, Vienna, Austria). Detection was carried out using SuperSignal^® Chemiluminescent Substrate (Thermo Fisher Scientific). Immunolabeling was visualized with the Fusion FX imaging system (Vilber Lourmat, Marne-la-Vallée, France) and band densitometry was performed using the Fusion© Software (Vilber Lourmat). Hsp90 and β-actin were used as housekeeping genes.

Immunofluorescence analyses were performed to localize and quantify changes in Nox1, Nox2 and Nox4. Kidney samples were fixed in 10% phosphate-buffered formalin in 0.1 M sodium phosphate-buffer (PB), cryoprotected in 30% sucrose in PB, after specimens were embedded in OCT, frozen in liquid nitrogen and stored at −80 °C. Nox1, Nox2 and Nox4 expression were determined by immunofluorescence. Slides were incubated with polyclonal primary antibodies: anti-Nox1, (ab55831), anti-Nox2 (PA-572816), antiNox4 (PA5-114489), from Abcam, Invitrogen and Thermoscientific, respectively, o/n at 4 °C. Then samples were incubated with Alexa Fluor 488 (Invitrogen) secondary antibody. After that, nuclei were stained with To-PRO (T-3605, Thermo-Fisher, Waltham, MA, USA). Slides were mounted with PBS/Glycerol.
Negative controls were processed identically but not primary antibody was used, Images of the specimens were collected with a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). The two channels were acquired sequentially to prevent crosstalk between them. The excitation and emission parameters used were (488 nm, 500–540 nm) for Nox1, Nox2 and Nox4 signal and (633 nm, 645–750 nm) for nuclei staining. The gains were adjusted for each channel to prevent saturation in pixels intensity.

The craniums collected from each group were meticulously trimmed using surgical instruments to remove the surrounding soft tissue and ensure thorough decalcification. Subsequently, the specimens were embedded and sectioned.
For H&E staining, the sections were baked at 60 °C for 40 min and then further dewaxed in xylene. After dewaxing, gradient ethanol dehydration was performed. Tissue sections were stained with hematoxylin and restained with eosin (Beijing Leagene Biotechnology, China). The stained sections were immersed in gradient ethanol, dried in a fume hood and sealed with resin. For TRAcP staining, TRAcP solution (Bizhong Bio, Suzhou) was prepared according to the manufacturer’s instructions. After the sections were dewaxed and dehydrated, they were incubated in a 37°C incubator for 1 hour. The slices were then washed with PBS for 5 min and soaked in xylene for 10 min. For immunohistochemistry, primary antibodies against NFATc1 (ab25916, Abcam) and NOX1 (ab55831, Abcam) were employed. Photographs were taken with an Axiovert 40 C optical microscope (Zeiss, Germany), and the positive cells in the area were counted and quantified by ImageJ software (Bethesda, USA).