Ab75941

Aβ_1-40/PBS-treated mouse RPE-choroid complex and incubated primary RPE cells were fixed with 4% paraformaldehyde. The analysis was performed as previously described 7 (link). Primary antibodies against the following were used for staining: 8-OHdG (1:100, sc-393871, Santa Cruz), cytochrome b245 light chain/p22^phox (1:500, ab75941, Abcam), and NOX4 (1:500, PA5-72816, Thermo Fisher Scientific).

Representative formalin-fixed paraffin-embedded tissue sections of 63 ovarian tumors in five epithelial ovarian cancer histopathologic types (HGSC, MUC, CCC, ENOC and undifferentiated carcinomas) and 30 normal tissues (ovarian, fallopian tube and endometrioid tissue) were prepared for evaluation of p22^phox protein expression by IHC, using an anti-p22^phox antibody with the following routine procedure. 5-μm sections were prepared. After dewaxing and hydration, sections were heated in citrate buffer in a microwave oven for 20 min for antigen retrieval. Anti-p22^phox rabbit polyclonal antibody (1:100, ab75941, Abcam Company, Cambridge, U.K.) was incubated with the sections overnight at 4 °C. The sections were then washed thrice in PBS for 3 min/wash and subsequently treated with a secondary antibody (ZSGB-BIO, Beijing, China) for one hour at 37 °C, followed by three washes in PBS for 3 min/wash. The p22^phox protein expression was visualized with diaminobenzidine (DAB) staining and visualized by light microscopy. A histological score (H-score) was constructed that measures the intensity and distribution of the signal using the formula: [3 (strong signal) × (percentage of cells with strong signal)] + [2 (moderate signal) × (percentage of cells with moderate signal)] + [1 (weak signal) × (percentage of cells with weak signal)]. The histological score ranges from 0 to 300.

The interaction of AOPPs with RANK and RAGE, p47^phox with p22^phox, Nox2 and Nox4, and phosphorylated p47^phox in cultured BMMs were determined by immunoprecipitation as described previously [21 (link)]. Antibody against AOPPs was kindly gifted from Dr. Jianwei Tian [33 (link)] (Southern Medical University, China). Antibody against RANK (sc-59981), RAGE (sc-365154) were from Santa Cruz (CA, USA). Antibodies against p47^phox (ab795), phosphor-p47^phox (ab166930), p22^phox (ab75941), NOX1 (ab131088), NOX4 (ab133303) were from Abcam (UK). Immune complexes were then tested by western blotting.

Mitotracker (red or far-red) and CellRox deep red were purchased from Thermo-Fischer and Invitrogen (Les Ulys, France), respectively; antibodies against NOX2 (ab80897) and p22^phox (ab75941) were acquired from Abcam; p47^phox (sc7660) and p67^phox (sc7663) were acquired from Santa Cruz; and NS1 was synthesized by Innoverda according to the published synthesis [19 (link)].

After each treatment, N2a cells were lysed in SDS buffer for 30 min. The protein assay kit (Beyotime Biotech, Shanghai, China) was used to evaluate the total protein concentrations. Cell lysate was separated with 10% SDS-PAGE and then transferred to PVDF membranes. Subsequently, the membranes were blocked and then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature and detection with an ECL kit (Thermo Scientific, USA). The antibodies are as follows: GPx-4 (1:2000, ab125066, Abcam), p22phox (1:1000, ab75941, Abcam), Mfn1 (1:1000, ab221661, Abcam), and GAPDH (1:5000, ab9485, Abcam).

Protein expression was analyzed by western blot using specific antibodies as described previously [21 (link)]. Antibodies against p38 (#8690), phosphor-p38 (#4511), ERK1/2 (#4695), phosphor-ERK1/2 (#4370), JNK (#9252), phosphor-JNK (#4668), c-fos (#4384), phosphor-c-fos (#5348), nuclear factor of activated T-cells (NFATc1, #8032), and GAPDH (#5174) were from Cell Signaling Technology (Beverly, USA); Antibodies against p47^phox (ab795), p22^phox (ab75941), NOX1 (ab131088), NOX4 (ab133303) were from Abcam (UK). Immunoreactive proteins were detected with horseradish peroxidase (HRP)-coupled secondary antibodies (goat anti rabbit, ab6721; rabbit anti goat, ab6741, Abcam, UK).