Journal: Endocrinology

Article Title: Periovulatory expression of hydrogen peroxide-induced sulfiredoxin and peroxiredoxin 2 in the rat ovary: gonadotropin regulation and potential modification.

doi: 10.1210/en.2012-1414

Figure Lengend Snippet: FIG. 6. Regulation of ovarian PRDX2 expression by gonadotropins. A, Aliquots of total RNA (20 g) isolated from ovaries at the indicated time intervals after eCG/hCG stimulation were assayed for PRDX2 mRNA levels by Northern blotting using a rat PRDX2 cDNA probe. The expression of 28S ribosomal RNA was used as an internal standard. The PRDX2 transcript was quantified using a phosphorimager and normalized for 28S RNA levels from three independently performed experiments (mean SEM). B, In situ localization of ovarian PRDX2 mRNA. Ovarian sections were hybridized with a 35S-labeled PRDX2 cRNA probe. Photomicrographs were taken under bright-field (a, c, and e) and corresponding dark-field (b, d, and f) illumination. Arrowhead, Oocyte; arrow, theca cell; asterisk, growing follicle; P, preovulatory follicle; CL, corpus luteum, 20. C, Western blotting analysis of PRDX2 protein in the ovary. Ovarian lysates were separated on a 12% SDS-PAGE gel in the presence (reducing) or absence (nonreducing) of 10% 2-mercaptoethanol. The quantified PRDX2 protein levels are expressed as a mean SEM of two or three independently performed experiments.

Article Snippet: Blots were incubated with primary antibodies against Srx, PRDX2, -PRDX-SO2/3, p-Akt (Cell Signaling Technology, Beverly, MA), Akt (Cell Signaling Technology), pPTEN (Cell Signaling Technology), or PTEN (Cell Signaling Technology).

Techniques: Expressing, Isolation, Northern Blot, In Situ, Labeling, Western Blot, SDS Page

Journal: Endocrinology

Article Title: Periovulatory expression of hydrogen peroxide-induced sulfiredoxin and peroxiredoxin 2 in the rat ovary: gonadotropin regulation and potential modification.

doi: 10.1210/en.2012-1414

Figure Lengend Snippet: FIG. 7. Modification of PRDX2 in preovulatory follicles after gonadotropin treatment. Preovulatory follicles were dissected from ovaries obtained at different times after eCG/hCG treatment. Cell lysates of preovulatory follicles were analyzed by SDS-PAGE (A) or two- dimensional PAGE (B) followed by immunoblot analysis with antibodies specific to Srx, sulfinic/sulfonic 2-Cys PRDXs (PRDX-SO2/3), PRDX2, and GAPDH. Lysates of HeLa cells exposed to 1 mM H2O2 for 10 min were used as a positive control for the expression of hyperoxidized 2-Cys PRDXs. The region on the two-dimensional immunoblots corresponds to a molecular mass of 22–28 kDa (vertical) and isoelectric points of 5.2–5.6 (horizontal). The band intensities of each sample were measured and the ratio of hyperoxidized (Ox) to reduced (Re) PRDX2 was used to plot the graph (B, lower panel). Each point on the graph represents mean SEM of three independently performed experiments. *, P 0.05 vs. 0 h.

Article Snippet: Blots were incubated with primary antibodies against Srx, PRDX2, -PRDX-SO2/3, p-Akt (Cell Signaling Technology, Beverly, MA), Akt (Cell Signaling Technology), pPTEN (Cell Signaling Technology), or PTEN (Cell Signaling Technology).

Techniques: Modification, SDS Page, Western Blot, Positive Control, Expressing