anti mbd3 14258 1 ap antibodies  (Proteintech)

Journal: Redox Biology

Article Title: Methylation reader MBD2-mediated GPX4 transcriptional repression drives ovarian granulosa cell ferroptosis in PCOS

doi: 10.1016/j.redox.2026.104034

Figure Lengend Snippet: GPX4 suppression is triggered by DNMT and MBD2-mediated Gpx4 promoter hypermethylation. (a) Schematic diagrams of the mouse Gpx4 promoter. The positions of the CpG island (gray area) and the methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) primers (boxes) are shown. (b) MSP analysis of the Gpx4 promoter in control (Ctrl) and DHEA-treated (DHEA) mice. (Left) Agarose gel images of MSP products from two randomly selected samples per group ( n = 6); (Right) Quantification of the methylated-to-unmethylated ratio, normalized to input. Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (c) BSP analysis of the Gpx4 promoter in Ctrl and DHEA mice. Quantification of the percentage of methylated CpG sites was on the right side. Data were presented as Box-and-whisker plots with data points. ∗ P < 0.05, Student's t -test. (d) Heatmap of normalized expression (FPKM) for 23 key transcription regulation-related genes from RNA-seq analysis of Ctrl and DHEA mice. The methylation-related methyltransferases DNMT1 and DNMT3a, significantly increase, and are highlighted in red. (e) Representative photomicrographs of ovarian sections from Ctrl and DHEA mice stained for DNMT1, DNMT3a and MBD2 by immunohistochemistry (IHC) staining. Positively-stained granulosa cells were indicated by arrows. (f) Western blot analysis of DNMT1, DNMT3a, DNMT3b, MBD2, MeCP2, MBD4, MBD3 and MBD1 proteins in ovarian tissues. GAPDH served as a loading control. Blots are representative of two samples per group. Quantification was presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (g) MSP analysis of the Gpx4 promoter in ovarian tissues from four treatment groups: Ctrl, 5-Azacytidine (5-Aza, 1.5 mg/kg, the last 10 days), DHEA (60 mg/kg, 21 days), and 5-Aza/DHEA. Agarose gel showing one representative sample per group. Right panel: Quantification of the methylated-to-unmethylated ratio, normalized to input. Data are presented as mean ± SEM, n = 6. ∗ P < 0.05, one-way ANOVA. (h) BSP analysis of the Gpx4 promoter. CpG methylation patterns from three mice per group (M1-M3), with five clones sequenced per mouse. Each row is a clone; each circle is a CpG site (open: unmethylated; closed: methylated). One box encompasses the data from one mouse. Quantification of the percentage of methylated CpG sites was on the right side. Data were presented as Box-and-whisker plots with data points. ∗ P < 0.05, one-way ANOVA.

Article Snippet: The following primary antibodies were used: GPX4 (ab125066, Abcam, UK), 4HNE (A26085, ABclonal, China), α-SMA (BS70000, Bioworld), Col1α (BS1530, Bioworld), DNMT1 (MA5-16169, Invitrogen, USA), DNMT3a (49768S, CST, USA), DNMT3b (48488S, CST, USA), MBD2 (ab188474, Abcam, UK), MeCP2 (A5694, ABclonal, China), MBD1 (29998-1-AP, Proteintech, USA), MBD3 (A8905, ABclonal, China), MBD4 (11270-1-AP, Proteintech, USA), FSHR (AF04761, AiFang Biological, China), Cyp19a1 (AF0449, AiFang Biological, China), MAZ (21068-1-AP, Proteintech, USA), NCoR (20018-1-AP, Proteintech, USA), HDAC3 (A19537, ABclonal, China), and GAPDH (FD0063, FDbio).

Techniques: Methylation, Methylation Sequencing, Control, Agarose Gel Electrophoresis, Whisker Assay, Expressing, RNA Sequencing, Staining, Immunohistochemistry, Western Blot, CpG Methylation Assay, Clone Assay