Fig. 7
Involvement of PI3K and STAT1 in the antioxidant effects of SOCS1 peptide. a Western blot analysis of cytosolic P-JAK2, P-PI3K and P-AKT (β-actin, loading control) and nuclear P-STAT1 and STAT1 in VSMC incubated with cytokines (103 U/mL IFNγ plus 102 U/mL IL6, 2 h) in the presence/absence of SOCS1 peptide (100 μg/mL). b Co-immunoprecipitation (IP) experiments of JAK2 and PI3K in cytokine-stimulated VSMC. c VSMC transfected with negative control scramble (scr) or specific siRNAs for PI3K (siPI3k) and STAT1 (siStat1) were stimulated for 2 h with cytokines with/without SOCS1 peptide. Lucigenin chemiluminescent assay was used to measure Nox-dependent O2•− production. Inset, PI3K and STAT1 immunoblotting in silenced cells from representative parallel transfection. d−f Lucigenin assay in MCT (d), VSMC (e) and macrophages (f) incubated with either cytokines (2 and 24 h) or HG (24 h) in the presence of PI3K inhibitors (wortmannin and LY294002), Nox inhibitors (apocynin and VAS2870), and SOCS1 peptide. g Immunodetection of Nox subunits in membrane fractions from VSMC at two different time points following cytokine stimulation. Representative immunoblots and summary of normalized densitometric quantification are shown. Data expressed as fold increases over basal conditions are the mean ± SEM of 3–7 experiments. *P < 0.05 vs Basal, #P < 0.05 vs Cytokines, and $P < 0.05 vs HG
