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

Journal: Antioxidants

Article Title: Tetraselmis chuii Supplementation Increases Skeletal Muscle Nuclear Factor Erythroid 2-Related Factor 2 and Antioxidant Enzyme Gene Expression, and Peak Oxygen Uptake in Healthy Adults: A Randomised Crossover Trial

doi: 10.3390/antiox14040435

Figure Lengend Snippet: Gene names, gene symbols and ThermoFisher Scientific Assay IDs for human skeletal muscle OpenArray™ gene expression analyses.

Article Snippet: Peroxiredoxin 2 , PRDX2 , Hs00853603_s1.

Techniques: Gene Expression, Binding Assay

Journal: Aging (Albany NY)

Article Title: Chetomin rescues pathogenic phenotype of LRRK2 mutation in drosophila

doi: 10.18632/aging.103843

Figure Lengend Snippet: LRRK2 interacts and phosphorylates PRDX2. ( A ) LRRK2-GFP and PRDX2-flag are co transfected in HEK293T cells. The lysates were collected and subjected to immunoprecipitation with anti-GFP antibody. The protein was subjected to western blot with anti-flag. Un-transfected cells, LRRK2 GFP alone and PRDX flag alone was transfected into HEK293T cells to serve as negative control. ( B ) Endogenous colocalization of LRRK2 (green) and PRDX2 (red) protein in SKNSH neuronal cells ( C ) In vitro kinase assay on SDS-PAGE gel show phosphorylation of PRDX2 by LRRK2 wildtype and G2019S.

Article Snippet: PRDX2 plasmid (RC207413) was purchased from Origene.

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, In Vitro, Kinase Assay, SDS Page

Journal: Aging (Albany NY)

Article Title: Chetomin rescues pathogenic phenotype of LRRK2 mutation in drosophila

doi: 10.18632/aging.103843

Figure Lengend Snippet: LRRK2 and PRDX2 transgenic flies. ( A ) LRRK2 GFP and PRDX2 expression driven by ddc-GAL-4 in transgenic fly head ( B ) Bar graphs show mean percentage and standard deviation of phosphorylation of PRDX2 in LRRK2 wildtype and G2019S flies (n = 3, cohort of 20).

Article Snippet: PRDX2 plasmid (RC207413) was purchased from Origene.

Techniques: Transgenic Assay, Expressing, Standard Deviation

Journal: Aging (Albany NY)

Article Title: Chetomin rescues pathogenic phenotype of LRRK2 mutation in drosophila

doi: 10.18632/aging.103843

Figure Lengend Snippet: PRDX2 is able to rescue G2019S pathogenic phenotype. ( A ) Representative magnified confocal images of whole mount brains 60 days after eclosion. The different clusters of TH+ neurons are boxed up and labeled. ( B ) Bar graphs show number of TH-positive DA neuron clusters in flies at 60 days after eclosion (n = 3, cohort of 10). ( C ) Bar graph shows climbing scores of male flies at 60 days after eclosion. Percentage of flies that reached the top of the column after 1 min was counted (n = 3, cohort of 20). ( D ) Bar graph shows number of flies that survive after 60 days. Age-matched ddc-GAL4/ + flies were used as controls. Percentage of flies was tabulated. Significance indicated on the graph: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: PRDX2 plasmid (RC207413) was purchased from Origene.

Techniques: Labeling

Journal: Physiological Reports

Article Title: Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation

doi: 10.14814/phy2.14745

Figure Lengend Snippet: Identification of L‐plastin and PRDXs in K562 cells. Conditioned media (CM) was collected and TCA precipitated, and the protein was extracted from cell lysates (CL) of K562 and MDA‐MB‐231 cells (MD‐231). a) Intracellular and released L‐plastin, PRDX2 and PRDX4 in K562 and MDA‐MB‐231 cells were assessed by immunoblotting. b) K562 cells were cultured at different cell densities (10 5 –10 6 cells/ml) and the levels of L‐plastin, PRDX2 and PRDX4 in CM were assessed by immunoblotting. Ponceau stain (lower gels) was used as loading control. Shown are representative immunoblots from one out five independent experiments.

Article Snippet: The following Taqman probes were used: L‐plastin, ( Lcp1 , Mm01310735_m1); Peroxiredoxin 2, ( Prdx2 , Mm04208213_m1); Matrix metalloprotease 9, ( Mmp9 , Mm00600163_m1); Trap ( Acp5 , Mm00475698_m1); erythroid transcription factor ( Gata ‐ 1 ; Mm00484679_g1); erythropoietin receptor ( Epor , Mm00438760_m1) and glyceraldehyde 3‐phosphate dehydrogenase ( Gapdh , Mm99999915_g1).

Techniques: Western Blot, Cell Culture, Staining, Control

Journal: Physiological Reports

Article Title: Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation

doi: 10.14814/phy2.14745

Figure Lengend Snippet: Acute anemia in mice increase the level of Prdx2 in bone marrow. Acute anemia was induced in female C57BL/6 mice by collecting 10% of total blood volume from saphenous vein, after which groups of animals were sacrificed at days 2, 3, 5 and 7. a‐b) RNA was isolated from bone marrow and gene expression for Prdx2 and Lcp1 was assessed. c‐d) Protein levels of Prdx2 and Lcp1 in bone marrow were assessed by immunoblotting. c) Representative immunoblots for Prdx2 ( top ), Lcp1 ( middle ) and Ponceau S‐stained gel ( bottom ) used for normalization. d) Average protein expression of Prdx2 and L‐plastin. For each immunoblot, protein signal was first normalized to ponceau staining, and then to the maximum signal within the immunoblot. Data are means ±SEM, N = 5–15 mice per condition, * p < 0.05 by one‐way ANOVA with Bonferroni post‐test, red : compared to control

Article Snippet: The following Taqman probes were used: L‐plastin, ( Lcp1 , Mm01310735_m1); Peroxiredoxin 2, ( Prdx2 , Mm04208213_m1); Matrix metalloprotease 9, ( Mmp9 , Mm00600163_m1); Trap ( Acp5 , Mm00475698_m1); erythroid transcription factor ( Gata ‐ 1 ; Mm00484679_g1); erythropoietin receptor ( Epor , Mm00438760_m1) and glyceraldehyde 3‐phosphate dehydrogenase ( Gapdh , Mm99999915_g1).

Techniques: Isolation, Gene Expression, Western Blot, Staining, Expressing, Control

Journal: Physiological Reports

Article Title: Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation

doi: 10.14814/phy2.14745

Figure Lengend Snippet: Exosomes from K562 cells induce osteoclast differentiation. a) RAW 264.7 cells were primed with RANKL (50 ng/ml) for 2 days and then cultured for an additional 2 days without RANKL treatment (negative control, NC), with RANKL (50 ng/ml, positive control, PC) or 10% CM from K562 cells +/− exosome inhibitor GW4869 (10 µM). N = 3. b) K562 cells were cultured for 24 h then exosomes were purified, and the distribution of particle sizes was analyzed by Nano sight. c) Representative transmission electron microscopy image of exosomes purified from K562 CM. d‐h) RAW 264.7 cells were primed with RANKL (50 ng/ml) for 2 days and then cultured for an additional 2 days without RANKL treatment (negative control, NC), with RANKL (50 ng/ml, positive control, PC) or with purified exosomes (Exo, 10 µl) from K562 cells. d) Representative images of TRAP‐stained osteoclasts formed in RAW cultures on day 5. Scale bar applies to all images. e) Average osteoclast number; f) average area per osteoclast; g) number of nuclei per osteoclast; h) average area/nucleus. N = 37–50 osteoclasts/condition; i) Immunoblotting for exosomal markers TFR‐2 and TSG101, L‐plastin, PRDX4 and PRDX2 in K562 CM and purified exosomes (Exo). Data are means ±SEM, * p < 0.05, ** p < 0.01 and *** p < 0.001 assessed by Student's t‐test; red : compared to negative control, blue : compared to ‐GW4869 or positive control.

Article Snippet: The following Taqman probes were used: L‐plastin, ( Lcp1 , Mm01310735_m1); Peroxiredoxin 2, ( Prdx2 , Mm04208213_m1); Matrix metalloprotease 9, ( Mmp9 , Mm00600163_m1); Trap ( Acp5 , Mm00475698_m1); erythroid transcription factor ( Gata ‐ 1 ; Mm00484679_g1); erythropoietin receptor ( Epor , Mm00438760_m1) and glyceraldehyde 3‐phosphate dehydrogenase ( Gapdh , Mm99999915_g1).

Techniques: Cell Culture, Negative Control, Positive Control, Purification, Transmission Assay, Electron Microscopy, Staining, Western Blot

Journal: Physiological Reports

Article Title: Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation

doi: 10.14814/phy2.14745

Figure Lengend Snippet: Recombinant Prdx2 induces osteoclast differentiation from RANKL‐primed precursors. RAW 264.7 cells were cultured for 4 days without RANKL (white bars), were treated with RANKL (50 ng/ml) for 4 days (black bars) or were RANKL‐primed (treated with RANKL 50 ng/ml for 2 days and cultured for an additional 2 days without RANKL, grey bars). a,b) Prdx2 at concentrations 0.5, 1, 2.5, or 5 µg/ml was added to untreated, RANKL‐treated and RANKL‐primed RAW 264.7. Shown are representative images of TRAP‐positive osteoclasts (a) and average number of osteoclasts formed in different conditions on day 4 (b). Scale bar applies to all images. Data are means ±SD, N = 2–3 replicates. c) Average number of osteoclasts formed when recombinant PRDX2 (2.5 or 5 µg/ml) was added to RANKL‐primed osteoclast precursors. Data are means ±SEM, N = 5–6 independent experiments, * p < 0.05 and ** p < 0.01 compared to RANKL‐primed cultures by Student's t‐test.

Article Snippet: The following Taqman probes were used: L‐plastin, ( Lcp1 , Mm01310735_m1); Peroxiredoxin 2, ( Prdx2 , Mm04208213_m1); Matrix metalloprotease 9, ( Mmp9 , Mm00600163_m1); Trap ( Acp5 , Mm00475698_m1); erythroid transcription factor ( Gata ‐ 1 ; Mm00484679_g1); erythropoietin receptor ( Epor , Mm00438760_m1) and glyceraldehyde 3‐phosphate dehydrogenase ( Gapdh , Mm99999915_g1).

Techniques: Recombinant, Cell Culture