Anti nf kb p65 phospho s536

Griess reagent, 5,5’-Dithiobis (2-nitrobenzoic acid) and Lucigenin were purchased from Sigma-Aldrich. Hydrogen peroxide 33% was procured from PanReac AppliChem. The following antibodies were used in this work: Anti-fibronectin was obtained from GeneTex. Anti-MMP-9 and anti-Bcl2 were obtained from Proteintech. Anti-vimentin, anti-MMP-3, Anti-Akt, Anti-p38, Anti-Cleaved PARP1, Anti-p53 (acetyl K382), Anti-JNK, Anti-alpha smooth muscle Actin, Anti-NF-kB p65 (phospho S536), and anti-β catenin were all obtained from Abcam (Cambridge, UK). Anti-cleaved caspase-3, anti-cleaved aspase-9, and anti-cleaved caspase-8 were obtained from Cell Signaling. Rabbit Polyclonal JNK1/2/3 was obtained from ORIGENE. Anti-beta Actin was obtained from Thermo Fisher Scientific. Peroxidase AffiniPure Goat Anti-Rabbit IgG and Peroxidase AffiniPure Goat Anti-Mouse IgG were obtained from Jackson ImmunoResearch Inc. The following ELISA kits were used in this work: nitrotyrosine ELISA kit (Abcam, ab113848), Hydrogen Peroxide Colorimetric/Fluorometric assay kit (Biovision, K265-200), malondialdehyde assay kits (Northwest Life Sciences Specialties, NWK-MDA01) and Superoxide Dismutase assay kit (Cayman chemical, 706002).

(i) Detection of exosome markers by Western blot. The exosomes obtained from the uterine cavity fluid were detected by Western blot for the purpose of the expression of CD9 (Anti‐CD9 antibody, Abcam, 1:1000 dilution, ab19761) protein in the exosomes. (ii) Detection of p65 (Anti‐NF‐kB p65 Monoclonal Antibody, Solarbio, 1:500, K200045M) and phosphorylation‐p65 (Anti‐NF‐kB p65 (Phospho S536), Abcam, 1:1000, ab86299) protein expression were operated as described previously (Wang, et al., 2019).

Endometrial epithelial cell, LPS‐treated EEC and LPS‐treated EEC with miR‐218 transfection were fixed in 4% paraformaldehyde for 30 min, and then permeabilized with 0.1% Triton X‐100 in PBS for 15 min, subsequently blocked with 5% BSA in PBS at room temperature for 1 h, and co‐incubated with anti‐ NF‐kB p65 (phospho S536) (Abcam, 1:1000, ab86299) at 37ºC for 2 h. After washing, it was incubated with secondary antibody at 37°C for 1 h, and the nuclei were stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI) for 3–5 min. The fluorescent signals were examined under a fluorescence microscope (Olympus).