Fig 1: Hypoxia–reoxygenation (H-R) challenge elicits a significant increase in the association and binding of NF-?B at the miR-210 proximal promoter. (A–C) miR-210 promoter pull-down assay determining the quantitative enrichment of the p65 NF-?B and p50 NF-?B subunits in the reverse crosslinked miR-210 promoter pull-down fragment. Representative Western blots (A) and quantitative densitometric analysis (B,C) showing the relative quantitative abundance of p65 NF-?B and p50 NF-?B subunits in the miR-210 promoter pull-down lysates. (D) Tandem ChIP-ELOHA analysis showing the relative enrichment of p65 NF-?B at the ?B response element in the miR-210 proximal promoter. Data from the Western blot-coupled densitometric analysis are expressed as mean fold change ± S.D. from three biological replicates belonging to each experimental group (n = 3). Data from the ChIP-ELOHA analysis are expressed as fold enrichment by first correcting the p65 NF-?B-associated miR-210 promoter fragment ELOHA absorbance values to the respective inputs, followed by normalization to fold change values. Data from the ChIP-ELOHA analysis are depicted as mean fold enrichment ± S.D. from three technical replicates for each of the four biological replicates belonging to each experimental group (n = 4). **** p = 0.0001. S.D.: standard deviation.
Fig 2: The activation and recruitment of NF-?B to the miR-210 proximal promoter is indispensable for the hypoxia–reoxygenation (H-R)-induced miR-210 expression. (A,B) Enzyme-coupled miR-210 hybridization immunoassay determining miR-210 levels in H-R challenge-subjected cells ectopically expressing either the da-I?Ba (A) or dn-IKKa/ß (B) mutants to inhibit NF-?B activation. (C,D) Tandem ChIP-ELOHA analysis showing the relative enrichment of p65 NF-?B at the ?B response element in the miR-210 proximal promoter in response to H-R challenge-subjected cells ectopically expressing either the da-I?Ba (C) or dn-IKKa/ß (D) mutants to inhibit NF-?B activation. Data from miR-210 hybridization immunoassay are expressed as mean fold change ± S.D. from three technical replicates for each of the four biological replicates belonging to each experimental group (n = 4). The ectopic expression of the HA-tagged da-I?Ba and HA-tagged dn-IKKa/ß mutants was validated using ELISA immunoassay performed against the HA tag (Supplementary Figure S3A–D). NF-?B transcriptional activity ((A–D), bottom panel) was determined in the native lysates to corroborate and validate the translative effects of the ectopic expression of the da-I?Ba and dn-IKKa/ß mutants. Data from the ChIP-ELOHA analysis are expressed as fold enrichment by first correcting the p65 NF-?B-associated miR-210 promoter fragment ELOHA absorbance values to the respective inputs, followed by normalization to fold change values. Data from the ChIP-ELOHA analysis are depicted as mean fold enrichment ± S.D. from three technical replicates for each of the four biological replicates belonging to each experimental group (n = 4). *** p = 0.001; **** p = 0.0001; ns: not significant (p > 0.05) S.D.: standard deviation.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for RelA/NFkB p65 NLS Antibody Blocking Peptide