Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Characterization of the sex-dependent myocardial S -nitrosothiol proteome

doi: 10.1152/ajpheart.00681.2015

Figure Lengend Snippet: S-nitrosoglutathione reductase (GSNO-R) expression and activity in male and female hearts. A: GSNO-R expression was assessed in male and female hearts via Western blot (n = 4 hearts/group; primary antibody: Origene, TA3211174, 1:1,000 dilution). B: GSNO-R activity was examined in male and female hearts (n = 11 hearts/group). Values are means ± SE. Male: solid bars; female: shaded bars. *P < 0.05.

Article Snippet: Membranes were blocked with 5% (wt/vol) nonfat dried milk or 5% (wt/vol) bovine serum albumin (for phospho-eNOS) in Tris-buffered saline with 0.1% Tween 20 for 1 h, and subsequently incubated with primary antibodies against eNOS (1:250; Santa Cruz Biotechnology, Dallas, TX), phospho-eNOS Ser1177 (1:1,000; Cell Signaling), nNOS (1:250; Santa Cruz Biotechnology), inducible NOS (iNOS) (1:250; Santa Cruz Biotechnology), GSNO-R (1:1,000; Origene, TA321117 4), sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) (1:1,000; Santa Cruz Biotechnology), aconitase (1:1,000; Cell Signaling, 6922), or GAPDH (1:1,000; Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Characterization of the sex-dependent myocardial S -nitrosothiol proteome

doi: 10.1152/ajpheart.00681.2015

Figure Lengend Snippet: Model of sex-dependent cardioprotection. Female hearts exhibit higher myocardial eNOS expression, phosphorylation (P), and NOx production, which facilitates enhanced SNO protein levels to induce a cardioprotective phenotype; enhanced GSNO-R activity in the female heart may also play a role in this pathway by preventing protein SNO from accumulating to levels that are sufficient to trigger nitrosative stress. Conversely, male hearts exhibit reduced myocardial eNOS expression, phosphorylation, and NOx production, thus leading to a level of protein SNO that falls below a critical threshold for cardioprotection; SNO protein levels can be increased in male hearts to facilitate cardioprotection with interventions, including ischemic preconditioning (IPC) (24, 47).

Article Snippet: Membranes were blocked with 5% (wt/vol) nonfat dried milk or 5% (wt/vol) bovine serum albumin (for phospho-eNOS) in Tris-buffered saline with 0.1% Tween 20 for 1 h, and subsequently incubated with primary antibodies against eNOS (1:250; Santa Cruz Biotechnology, Dallas, TX), phospho-eNOS Ser1177 (1:1,000; Cell Signaling), nNOS (1:250; Santa Cruz Biotechnology), inducible NOS (iNOS) (1:250; Santa Cruz Biotechnology), GSNO-R (1:1,000; Origene, TA321117 4), sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) (1:1,000; Santa Cruz Biotechnology), aconitase (1:1,000; Cell Signaling, 6922), or GAPDH (1:1,000; Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay

Journal: EBioMedicine

Article Title: Deiodinase-3 is a thyrostat to regulate podocyte homeostasis

doi: 10.1016/j.ebiom.2021.103617

Figure Lengend Snippet: Activation of TSH-R on podocytes is detrimental. (a) qPCR data showing the average Ct value from 2 independent wells for tshr gene in mouse podocytes and mouse thyroid in comparison to the housekeeping gene gapdh . (b) Western blot analysis of lysates from mouse podocytes, mouse thyroid tissue (positive control) and human K562 cells (negative control) probed with the indicated antibodies. Representative blot from two independent experiments is shown. Migration of molecular weight standards is depicted on the left as kDa. (c) Histogram depicting the data from flow cytometric analysis performed on mouse podocytes with FITC-conjugated TSH-R antibody (blue) overlapped with the controls (unstained, red and FITC-conjugated IgG2a isotype control, green). 5000 events were collected and gated. Graph on the right shows the geometric mean fluorescence intensity (MFI) on Y-axis obtained from four independent experiments. Non-parametric two-tailed Students t -test was used to calculate the significance, * p≤0.05 . (d) Confocal micrographs of cultured mouse podocytes left untreated (UT), PAN treated (30 µg/ml), Normal rabbit IgG isotype or anti-TSH-R antibody (3 µg/ml each) for 24 h. Stains: merge of F-actin phalloidin-488 stained in green and DAPI in blue. Scale bar=20 µm. (e) Confocal micrographs of cultured mouse podocytes left untreated (UT), PAN treated (30 µg/ml), or TSH (25 and 50 nM) for 24 h. Stains: merge of F-actin phalloidin-488 in green and DAPI in blue. Scale bar=20 µm. (f) Quantification of the data in d, represented as percentage of cells with normal cell shape and size with transversal stress fibers. Non-parametric two-tailed Students t -test was used to calculate the significance, ** p≤0.01 and *** p≤0.001 . (g) Quantification of the data in e, represented as percentage of cells with normal cell shape and size with transversal stress fibers. Non-parametric two-tailed Students t -test was used to calculate the significance, *** p≤0.001 . (h) cAMP levels (in pmol/ml) normalized with the protein concentration (mg/ml), plotted as pmol/mg in the culture supernatants of mouse podocytes exposed to the indicated treatments for 60 min. Non-parametric two-tailed Students t -test was used to calculate the significance, *** p≤0.001 and * p≤0.05 . (i) qPCR analysis of DIO3 transcript in mouse podocytes upon PAN (30 µg/ml) and TSH (25 and 50 nM) treatment for 24 h. gapdh was used as the housekeeping gene; ** p≤0.01 and * p≤0.05 determined using non-parametric Students t -test.

Article Snippet: In a final volume of 50 µl containing 2 × 10 5 cells, staining was performed with 1:50 dilution of either Rabbit anti-TSH-R-FITC conjugated antibody (Bioss Antibodies, BS-0460R-FITC; RRID:AB_11042712) or normal mouse IgG-FITC (Santa Cruz, sc-2856; RRID:AB_737238) for 1 h at 4°C.

Techniques: Activation Assay, Western Blot, Positive Control, Negative Control, Migration, Molecular Weight, Fluorescence, Two Tailed Test, Cell Culture, Staining, Protein Concentration