nox2 polyclonal antibody  (Proteintech)

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate Suppresses NOX2-Derived ROS to Regulate Macrophage Function and Lesion Progression in End ometriosis (A-B) qPCR analysis of NOX2 mRNA in peritoneal macrophages (PMs) from human (A) and mouse (B) NC and EM groups (n = 3/group). (C-D) Western blot and quantification of NOX2 protein in PBMC-derived macrophages treated with LPS or LPS + 4-OI (n = 5/group). (E) NOX2 enzyme activity in BMDMs (n = 4/group). (F, G) Flow cytometry and quantification of ROS production in BMDMs after LPS or LPS + 4-OI (n = 3/group). (H, I) Flow cytometry and quantification of iNOS + BMDMs after LPS, LPS + 4-OI, or LPS + 4-OI + DPI treatment (n = 4/group). (J) Intracellular Ca 2+ dynamics in BMDMs measured by Fluo-4 after LPS, 4-OI, or DPI treatment. (K) Schematic of 4-OI and DPI intervention in the mouse endometriosis model. (L, M) Representative images (L) and gross morphology (M) of endometriotic lesions after PBS, 4-OI, or DPI treatment. (N) Quantification of lesion weight (n = 6/group). (O, P) Flow cytometry and quantification of iNOS + peritoneal macrophages (percentage and MFI) in peritoneal lavage (n = 6/group). (Q) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in peritoneal macrophages after treatments (n = 6/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, ns: not significant.) E2, estradiol benzoate; EM, endometriosis; NC, non-EM control; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; DPI, NOX2 inhibitor.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Derivative Assay, Western Blot, Activity Assay, Flow Cytometry, Control

Journal: Redox Biology

Article Title: Stromal cell-derived itaconate promotes endometriosis via macrophage NRF2 and lysosomal pH modulation

doi: 10.1016/j.redox.2026.104101

Figure Lengend Snippet: Itaconate Regulates Lysosomal Function, Calcium Signaling, and p38 MAPK Pathway in Macrophages (A) Lysosomal acidification in ascites-derived macrophages from non-endometriosis (NC) and endometriosis (EM) patients assessed by LysoSensor fluorescence intensity (n = 6/group). (B) Lysosomal acidification in PBMC-derived macrophages after LPS,IL-4 and 4-OI treatment (n = 3/group). (C) Lysosomal pH value of BMDMs after LPS and 4-OI treatment (n = 3/group). (D, E) Effect of chloroquine (CQ) on LysoSensor intensity (D) and proportion of iNOS + BMDMs (E) after LPS and 4-OI treatment (n = 3/group). (F) mRNA levels of Il1b , Il6 , Nos2 , and Tnf in BMDMs under indicated treatments (n = 3/group). (G) Intracellular Ca 2+ dynamics in BMDMs after treatments, measured by Fluo-4. (H) Intracellular Ca 2+ dynamics in ascites-derived macrophages from NC and EM patients. (I) Quantification of intracellular calcium in peritoneal macrophages from NC and EM patients (n = 3/group). (J) Quantification of intracellular calcium in BMDMs (n = 5/group). (K) mRNA expression of lysosomal calcium channel genes ( MCOLN1 , MCOLN2 , TPC1 , TPC2 ) in ascites-derived macrophages from NC and EM patients (n = 3/group). (L) lysosomal calcium channel genes mRNA in PBMC-derived macrophages co-cultured with normal ESCs (Nor-ESC), eutopic ESCs (Eu-ESC), or ectopic ESCs (Ec-ESC) from patients (n = 5/group). (M − N) Fluo-4-based Ca 2+ dynamics in BMDMs treated with LPS, 4-OI, ML-SA1 (M), or CQ (N). (O) Flow cytometry analysis and quantification of iNOS + BMDMs after indicated treatments (n = 3/group). (P) mRNA levels of pro-inflammatory genes in BMDMs under different treatments (n = 3/group). (Q) Western blot and quantification of p-p38 and total p38 in BMDMs with LPS ± 4-OI (n = 3/group). (R–S) Western blot and quantification of iNOS, p-p38, and total p38 in BMDMs treated with LPS, 4-OI, and CQ (R), or ML-SA1 (S) (n = 3/group). (Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) EM, endometriosis; NC, non-EM control; LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; CQ, chloroquine; ML-SA1, MCOLN channel agonist; PBMC, peripheral blood mononuclear cell; ESC, endometrial stromal cell.

Article Snippet: After blocking in 5% milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies: Beta Actin Monoclonal antibody(1:20000, 66009-1-Ig; Proteintech), Beta Tubulin Recombinant antibody (1:5000, 80713-1-RR; Proteintech), Anti-IRG1 antibody (1:1000; ab222411; abcam; Cambridge, UK), Nrf2 monoclonal antibody (1:2000; A21176; abclonal; Wuhan, China), iNOS Polyclonal antibody (1:500; 22226-1-AP; Proteintech), NOX2 Polyclonal antibody (1:3000; 19013-1-AP; Proteintech), p-p38 MAPK Polyclonal antibody (1:2000; 28796-1-AP; Proteintech), and p38 MAPK Polyclonal antibody (1:4000; 14064-1-AP; Proteintech).

Techniques: Derivative Assay, Fluorescence, Expressing, Cell Culture, Flow Cytometry, Western Blot, Control