Fig 1: Combinations of AR and ASR at 3:1 and 5:1 increase border zone capillary density in hearts of mice at 28 days after MI. (A) Representative immunofluorescence images taken within the infarct border zone of mice treated with saline, or different combinations of AR and ASR. Capillaries were stained with CD31, nuclei were stained with DAPI. Scale bar = 50 μm. (B) Graph depicting the quantification of border zone capillary number across treatments presented as the number of CD31-positive capillaries per high-power field (HPF). n = 6 in each group. Values are expressed in mean ± S.D. #p < 0.05, ##p < 0.01 vs. vehicle mice treated with saline. (C, D) Representative example and quantification of VEGF, p-Akt and p-eNOS protein levels (Western blotting) in the myocardial tissue at 28 days after MI surgery and treatment with different combinations of AR and ASR or saline. n = 4 in each group. Values are expressed in mean ± S.D. *p < 0.05 vs. sham mice treated with saline, #p < 0.05, ##p < 0.01 vs. vehicle mice treated with saline.
Fig 2: GATA4 mediates NOX4 expression. (A) GATA1-6 mRNA expression in HUVECs exposed to control or hyperglycemic conditions; ***P<0.001, hyperglycemia vs. control group. (B) Chromatin immunoprecipitation results for binding of GATA4 to the NOX4 promoter under normoglycemic control conditions and hyperglycemic conditions; %%%P<0.001, control anti-Flag vs. control anti-IgG group; ##P<0.01, hyperglycemia anti-Flag vs. control anti-Flag group. (C) NOX4 mRNA expression upon GATA4 overexpression; ***P<0.001, GATA4 overexpression group vs. control. (D) NOX4 and p-eNOS protein expression in HUVECs overexpressing GATA4 were measured by western blot analysis; **P<0.01, GATA4 overexpression group vs. control; &&&P<0.001, GATA4 overexpression group vs. control. eNOS, endothelial nitric oxide synthase; GATA, GATA-binding protein; IgG, immunoglobulin G; NOX4, NADPH oxidase 4; p, phosphorylated.
Fig 3: Agrin does not regulate endothelial ERK1/2 mediated activation in vivo. (A) Western blotting analysis showing no significant difference in endothelial expression and activation of ERK1/2 or eNOS after endothelial loss of agrin in vivo. The expression and activation levels of (B,C) ERK1/2, (D,E) Akt and (F,G) eNOS; as well as the expression levels of (H) VEGF-A were measured in the skeletal muscle ECs of 6–8 weeks old EC-agrn+/+ and EC-agrncKO mice, respectively. (B–H) Data are presented as mean ± S.E.M., n = 3, differences were determined by ANOVA and Turkey's HSD post-hoc test, **P < 0.01, n.s. denotes no significant difference.
Fig 4: pGz activates eNOS in dystrophic myotubes.A. Myotubes were treated with pGz (120 cpm, 12 h) for the indicated times, quickly lysed in modified RIPA buffer and eNOS phosphorylation (Ser632) was assessed by western blot. B. L-NAME treatment blocks the pGz-induced reduction of [Ca2+]i in mdx myotubes. Data are expressed as mean ± S.E.M. *P<0.05, **P<0.01, ***P<0.001, ANOVA-Dunnett’s (A) and Tukey’s. (B).
Fig 5: nNOS and eNOS protein levels in muscles from pGz-treated mice.Gastrocnemius were dissected, homogenized and protein expression was assessed by western blot. A. Representative western blot. B. Quantification of eNOS and C. nNOS. Each lane in the western blot represents a muscle sample obtained from a different mouse. Data were normalized with GAPDH. Data are expressed as mean ± S.E.M. from N mice, *P<0.05, and ***P<0.001, ANOVA-Tukey’s.
Supplier Page from Abcam for Anti-eNOS (phospho S632) antibody [M232]