p22 phox  (Santa Cruz Biotechnology)

Journal: Biomolecules & Therapeutics

Article Title: γ-Oryzanol Ameliorates Endothelial Replicative Senescence via Downregulation of SGLT2 Expression to Attenuate NADPH-Driven Oxidative Stress

doi: 10.4062/biomolther.2025.133

Figure Lengend Snippet: γ-Orz downregulates SGLT2 expression and reduces NADPH oxidase-induced oxidative stress. (A) Representative blots of SGLT2, p22 phox , NOX2, and eNOS in P3 ECs in presence of γ-Orz (3, 10 µM). (B-E) Cumulative data for expression of SGLT2, p22 phox , NOX2, and eNOS respectively. Data are expressed as mean ± SEM (n=3). # p <0.05, ## p <0.01 vs. P1; * p <0.05; ** p <0.01 vs. P3.

Article Snippet: The protein was loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel and separated by electrophoresis, then transferred to a polyvinylidene difluoride membrane that was blocked with 5% bovine serum albumin (BSA) for 1 h and incubated overnight at 4°C with primary antibodies against β-actin (sc-47778, 1:5000; Santa Cruz Biotechnology); p53 (sc-6243, 1:1000; Santa Cruz Biotechnology); p21 (sc-397, 1:1000; Santa Cruz Biotechnology); p16 (sc-1661, 1:1000; Santa Cruz Biotechnology); p22 phox (sc-271968, 1:1000; Santa Cruz Biotechnology); SGLT2 (AGT-032, 1:1000, Alomone labs); eNOS (cat. no. 610297, 1:1000; BD biosciences); NOX2 (cat. no. 19013-1-AP, 1:1000; Protein tech); diluted in 5% BSA.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: NOX4-derived oxidative DNA damage impairs thyroid differentiation through an epigenetic mechanism in BRAF-mutated radioactive iodine refractory papillary thyroid cancer cells

doi: 10.7150/ijbs.123980

Figure Lengend Snippet: Knockdown of NOX4 or p22 phox reduces oxidative-DNA damage. A) Western-blot analysis of NOX4 protein expression in nuclear fractions of thyrocytes and BRAF-mutated thyroid cells (n=2) B) Quantification of 8-oxoG of BRAF-mutated thyroid cells by FACS analysis of immune-stained 8-oxoG cells. The cells were transduced with siRNA control or siRNA against NOX4 or p22 phox for 72 h. Cells were incubated with an anti-8oxoG or a mouse IgG2B (isotype control). Graphs show the quantification of the fluorescence mean. Values are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3). C) Genomic distribution of γH2AX in BCPAP cells depleted or not for NOX4 (72h). Two replicates for each condition (siCtrl. and siNOX4) were pooled. D) Distribution of γH2AX on genome for BCPAP cells depleted or not for NOX4. The x axis corresponds to the position of the peaks along chromosomes; the y axis corresponds to the MACS2 score (-10*(log10(q_value)). E) γH2AX enrichment peaks on selected genes in conditions siRNA control and siRNA NOX4.

Article Snippet: The membranes were probed with primary antibodies: anti-NOX4 (Abcam, #ab109225, RRID:AB_10861375); anti-p22 phox (Santa Cruz Biotechnology, #Sc-130551, RRID:AB_2245805); anti-LaminB1 (Abcam, #ab133741, RRID:AB_2616597); anti-MSH6 (Protein Tech, #A3000 22 A); anti-MSH2 (Abcam, #ab70270); anti-OGG1/2 (G5) (Santa Cruz Biotechnology, #Sc376935); anti-Vinculin (Abcam, #ab130007); anti-DNMT1 (Abcam, #ab13537); anti-DNMT3a (Abcam, #ab2850); anti-DNMT3b (Cell signaling, #695202); anti-PAX8 (Cell signaling, #59019); anti-TTF1 (NKX2.1) (Cell signaling, #123735), anti phospho-p44/42 ERK (Cell signaling, #4370); anti-ERK (Cell signaling, #4696); anti phosphor-MEK1/2 (Cell signaling, #9121); anti-MEK (Cell signaling, #4694); anti-BRAF V600E (Spring, #E19290); anti-phospho-Smad3 (Abcam, #ab515663); anti-Smad3 (Thermo Scientific, # MA515663 ) or anti-Myc-Tag (Cell signaling, #22765) overnight at 4 °C with constant agitation.

Techniques: Knockdown, Western Blot, Expressing, Staining, Transduction, Control, Incubation, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: NOX4-derived oxidative DNA damage impairs thyroid differentiation through an epigenetic mechanism in BRAF-mutated radioactive iodine refractory papillary thyroid cancer cells

doi: 10.7150/ijbs.123980

Figure Lengend Snippet: Knockdown of NOX4 or p22 phox reduces the recruitment of OGG1, MSH2, and MSH6 to chromatin. A) Western-blot analysis of MSH6, MSH2, and OGG1 protein expression levels in cytosolic and chromatin fractions of BRAF V600E -mutated thyroid cells (n=2). B) Western blot analysis of NOX4 and p22 phox protein expression levels in nuclear fractions 72 h after knocking down of NOX4 or p22 phox by RNA interference in BCPAP cells. C) Western blot analysis of MSH6, MSH2, and OGG1 protein expression levels in chromatin fractions and whole-cell extracts 72 h after knockdown of NOX4 or p22 phox by RNA interference in BCPAP cells. Densitometry quantification of protein levels normalized to Lamin B or Vinculin levels and presented as chromatin enrichment or fold change compared with control cells. Values are mean ± SE. *p < 0.05, ***p < 0.001 and ****p<0.0001 (n = 3).

Article Snippet: The membranes were probed with primary antibodies: anti-NOX4 (Abcam, #ab109225, RRID:AB_10861375); anti-p22 phox (Santa Cruz Biotechnology, #Sc-130551, RRID:AB_2245805); anti-LaminB1 (Abcam, #ab133741, RRID:AB_2616597); anti-MSH6 (Protein Tech, #A3000 22 A); anti-MSH2 (Abcam, #ab70270); anti-OGG1/2 (G5) (Santa Cruz Biotechnology, #Sc376935); anti-Vinculin (Abcam, #ab130007); anti-DNMT1 (Abcam, #ab13537); anti-DNMT3a (Abcam, #ab2850); anti-DNMT3b (Cell signaling, #695202); anti-PAX8 (Cell signaling, #59019); anti-TTF1 (NKX2.1) (Cell signaling, #123735), anti phospho-p44/42 ERK (Cell signaling, #4370); anti-ERK (Cell signaling, #4696); anti phosphor-MEK1/2 (Cell signaling, #9121); anti-MEK (Cell signaling, #4694); anti-BRAF V600E (Spring, #E19290); anti-phospho-Smad3 (Abcam, #ab515663); anti-Smad3 (Thermo Scientific, # MA515663 ) or anti-Myc-Tag (Cell signaling, #22765) overnight at 4 °C with constant agitation.

Techniques: Knockdown, Western Blot, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: NOX4-derived oxidative DNA damage impairs thyroid differentiation through an epigenetic mechanism in BRAF-mutated radioactive iodine refractory papillary thyroid cancer cells

doi: 10.7150/ijbs.123980

Figure Lengend Snippet: Knockdown of NOX4 or p22 phox as well as DNA repair proteins reduce the recruitment of DNMT1 to chromatin. A) Western-blot analysis of DNMT1 in chromatin fraction of thyrocytes and BRAF V600E -mutated thyroid cells (n=2). B) Western blot analysis of DNMT1 protein expression in chromatin fractions and whole-cell extracts 72 h after knocking down of NOX4 or p22 phox by RNA interference in BCPAP cells. C) Western blot analysis of DNMT1 protein expression in chromatin fractions and whole-cell extracts 72 h after knocking down of OGG1 by RNA interference in BCPAP cells. D) Western blot analysis of DNMT1 protein expression in chromatin fractions and whole-cell extracts 48 h after knocking down of MSH2 by RNA interference in BCPAP cells. Densitometry quantification of protein levels normalized to Lamin B or Vinculin levels and presented as chromatin enrichment or fold change compared with control cells. Values are mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p<0.0001 (n = 3).

Article Snippet: The membranes were probed with primary antibodies: anti-NOX4 (Abcam, #ab109225, RRID:AB_10861375); anti-p22 phox (Santa Cruz Biotechnology, #Sc-130551, RRID:AB_2245805); anti-LaminB1 (Abcam, #ab133741, RRID:AB_2616597); anti-MSH6 (Protein Tech, #A3000 22 A); anti-MSH2 (Abcam, #ab70270); anti-OGG1/2 (G5) (Santa Cruz Biotechnology, #Sc376935); anti-Vinculin (Abcam, #ab130007); anti-DNMT1 (Abcam, #ab13537); anti-DNMT3a (Abcam, #ab2850); anti-DNMT3b (Cell signaling, #695202); anti-PAX8 (Cell signaling, #59019); anti-TTF1 (NKX2.1) (Cell signaling, #123735), anti phospho-p44/42 ERK (Cell signaling, #4370); anti-ERK (Cell signaling, #4696); anti phosphor-MEK1/2 (Cell signaling, #9121); anti-MEK (Cell signaling, #4694); anti-BRAF V600E (Spring, #E19290); anti-phospho-Smad3 (Abcam, #ab515663); anti-Smad3 (Thermo Scientific, # MA515663 ) or anti-Myc-Tag (Cell signaling, #22765) overnight at 4 °C with constant agitation.

Techniques: Knockdown, Western Blot, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: NOX4-derived oxidative DNA damage impairs thyroid differentiation through an epigenetic mechanism in BRAF-mutated radioactive iodine refractory papillary thyroid cancer cells

doi: 10.7150/ijbs.123980

Figure Lengend Snippet: DNMT inhibition and knockdown of MSH2, MSH6, and OGG1 synergize to increase NIS mRNA expression. A) qRT-PCR analysis of NIS mRNA levels in BCPAP cells transfected with siRNA control or/and siRNA MSH2 or/and siRNA MSH6 or/and siRNA OGG1 and 24 h later treated for an additional 48 h in the presence or the absence of 1 µM DAC. B) qRT-PCR analysis of NIS mRNA levels in BCPAP cells transfected with siRNA control or siRNA NOX4 or siRNA p22 phox and 24 h later, treated for an additional 48 h in the presence or the absence of 1 µM DAC. Values are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001. C) Western blot analysis of PAX8 and NKX2.1 protein expressions in chromatin fraction and whole-cell extract 72 h after knocking down of NOX4 or p22 phox by RNA interference in BCPAP cells. Densitometry quantification of protein levels normalized to Lamin B or Vinculin levels and presented as chromatin enrichment or fold change compared with control cells. Values are mean ± SE. *p < 0.05 and **p < 0.01 (n = 5). D) ChIP-qPCR assays performed with BCPAP cells transfected with siRNA control and with BCPAP cells transfected with siRNA NOX4 immunoprecipitated with control IgG or anti-NKX2.1 antibody and analyzed by qPCR at TG promoter (two independent replicates).

Article Snippet: The membranes were probed with primary antibodies: anti-NOX4 (Abcam, #ab109225, RRID:AB_10861375); anti-p22 phox (Santa Cruz Biotechnology, #Sc-130551, RRID:AB_2245805); anti-LaminB1 (Abcam, #ab133741, RRID:AB_2616597); anti-MSH6 (Protein Tech, #A3000 22 A); anti-MSH2 (Abcam, #ab70270); anti-OGG1/2 (G5) (Santa Cruz Biotechnology, #Sc376935); anti-Vinculin (Abcam, #ab130007); anti-DNMT1 (Abcam, #ab13537); anti-DNMT3a (Abcam, #ab2850); anti-DNMT3b (Cell signaling, #695202); anti-PAX8 (Cell signaling, #59019); anti-TTF1 (NKX2.1) (Cell signaling, #123735), anti phospho-p44/42 ERK (Cell signaling, #4370); anti-ERK (Cell signaling, #4696); anti phosphor-MEK1/2 (Cell signaling, #9121); anti-MEK (Cell signaling, #4694); anti-BRAF V600E (Spring, #E19290); anti-phospho-Smad3 (Abcam, #ab515663); anti-Smad3 (Thermo Scientific, # MA515663 ) or anti-Myc-Tag (Cell signaling, #22765) overnight at 4 °C with constant agitation.

Techniques: Inhibition, Knockdown, Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, ChIP-qPCR, Immunoprecipitation

Journal: International Journal of Biological Sciences

Article Title: NOX4-derived oxidative DNA damage impairs thyroid differentiation through an epigenetic mechanism in BRAF-mutated radioactive iodine refractory papillary thyroid cancer cells

doi: 10.7150/ijbs.123980

Figure Lengend Snippet: MAPK pathway inhibition and knockdown of NOX4 and p22 phox synergize to promote PAX8 and NKX2.1 recruitment to chromatin. A) Western-blot analysis of PAX8 and NKX2.1 protein expressions in chromatin fractions and whole-cell extracts of BCPAP cells transduced with siRNA control or siRNA NOX4 or siRNA p22 phox and treated with dabrafenib (100 nM) plus trametinib (25 nM) combination for 48 h. Densitometric quantification of protein levels normalized to Lamin B or loading control (red ponceau staining) and presented as chromatin enrichment and fold change compared with siRNA control-transduced cells. B) qRT-PCR analysis of NIS mRNA levels in BCPAP cells transfected with siRNA control or siRNA NOX4 or siRNA p22 phox and 24 h later, treated in the presence or the absence of dabrafenib plus trametinib combination for additional 48 h. C) qRT-PCR analysis of TSHR mRNA levels in BCPAP cells transfected with siRNA control or siRNA NOX4 or siRNA p22 phox and 24 h later, treated in the presence or the absence of dabrafenib plus trametinib combination for additional 48 h. Values are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3).

Article Snippet: The membranes were probed with primary antibodies: anti-NOX4 (Abcam, #ab109225, RRID:AB_10861375); anti-p22 phox (Santa Cruz Biotechnology, #Sc-130551, RRID:AB_2245805); anti-LaminB1 (Abcam, #ab133741, RRID:AB_2616597); anti-MSH6 (Protein Tech, #A3000 22 A); anti-MSH2 (Abcam, #ab70270); anti-OGG1/2 (G5) (Santa Cruz Biotechnology, #Sc376935); anti-Vinculin (Abcam, #ab130007); anti-DNMT1 (Abcam, #ab13537); anti-DNMT3a (Abcam, #ab2850); anti-DNMT3b (Cell signaling, #695202); anti-PAX8 (Cell signaling, #59019); anti-TTF1 (NKX2.1) (Cell signaling, #123735), anti phospho-p44/42 ERK (Cell signaling, #4370); anti-ERK (Cell signaling, #4696); anti phosphor-MEK1/2 (Cell signaling, #9121); anti-MEK (Cell signaling, #4694); anti-BRAF V600E (Spring, #E19290); anti-phospho-Smad3 (Abcam, #ab515663); anti-Smad3 (Thermo Scientific, # MA515663 ) or anti-Myc-Tag (Cell signaling, #22765) overnight at 4 °C with constant agitation.

Techniques: Inhibition, Knockdown, Western Blot, Transduction, Control, Staining, Quantitative RT-PCR, Transfection

Journal: bioRxiv

Article Title: Dysregulation of U12-Type Splicing in Lupus Neutrophils

doi: 10.1101/2025.11.06.686965

Figure Lengend Snippet: A) Western blot (upper and middle and respective densitometry analysis (lower) showing the expression of CYBA (P22phox) and myeloperoxidase (MPO, as a house keeping protein) in neutrophils from healthy controls (n=6), SLE (n=11) or CGD (n=2) patients. The top Western gel compares LDGs with autologous NDGs whereas the bottom Western represents NDGs only; * p<0.05. B) Nox activity measured after stimulation with either fMLF (upper graph) or PMA (lower graph) in neutrophils obtained from SLE patients (n=7) or healthy controls (n=12). Asterisks in fMLF indicate statistically significant difference between lupus LDG and NDG, and in PMA significant difference between LDG and healthy control NDG; (*p<0.05), applied Mann-Whitney U-test. C) Images of neutrophils stained with an antibody against P22phox and a secondary antibody (FITC conjugated, green). Nuclei are stained blue with Hoechst (magnification 40x). D) Expression of P22phox in neutrophils measured by flow cytometry. Results represent mean + SEM. Of SLE patients whose samples were tested for CYBA expression and activity, 83% were anti-ANA and/or anti-ENA positive (50% anti-Ro, 33% anti-Sm, 66% anti-RNP, none anti-La) and mean SLEDAI was 3.6 + 3.

Article Snippet: The cells were then stained with anti-P22phox antibody or isotype control antibody (Santa Cruz Biotechnology), followed by staining with fluorescent-conjugated secondary antibodies.

Techniques: Western Blot, Expressing, Activity Assay, Control, MANN-WHITNEY, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Renoprotective effects of GHRH agonist MR409 is associated with reduced oxidative stress and ferroptosis in diabetic mice

doi: 10.3389/fphar.2025.1617185

Figure Lengend Snippet: MR409 attenuates oxidative stress in the kidney of db/db mice. (A) Representative images of ROS oxidative fluorescence intensity in the kidney of db/db mice stained by DHE. (B) Quantitative analysis of the relative fluorescence intensity. (C,D) Protein expressions of p22phox and gp91phox. Data are expression as mean ± SEM. ** P < 0.01 vs control (ctr) group; ## P < 0.01, # P < 0.05 vs db/db group. n = 6. Scale bar = 250 μm.

Article Snippet: Following blocking, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-p22phox (sc-271262), anti-Fibronectin (sc-271098), and anti-TGFβ1 (sc-146) from Santa Cruz Biotechnology; anti-phospho-endothelial nitric oxide synthase (eNOS at Ser1177, AF3247) from Affinity Biosciences; anti-peroxisome proliferator-activated receptors γ (PPARγ, #2435) from Cell Signaling Technology; anti-gp91phox (ab80508), anti-GHRHR (ab76263) and anti-Klotho (ab181373) from Abcam; and anti-β-actin (AC004) from AB clonal.

Techniques: Fluorescence, Staining, Expressing, Control