ONOO⁻ promotes 26S proteasome assembly both <i>in vitro</i> and in intact cell.

<p><i>In vitro</i> (A): ONOO⁻ (1 µM) was incubated with the purified 26S proteasome for 5 min; in intact cell (B): HUVEC was incubated with ONOO⁻ for 0.5 h, in the presence or absence of uric acid (50 µM pre-incubation for 1 h). Cell free system (<i>in vitro</i>) was subjected to (A) separation on a native gradient (3–14%) PAGE gel followed either by Western-blotting (IB) or a direct staining with coomassie brilliant blue (CBB staining) for 26S proteasome assembly. HUVEC cell lysate was subjected to (B) Western blotting of the PA700/S10B co-immunoprecipitates with a β7 antibody. All blots shown are representative of three independent experiments. All results (n = 3) were analyzed with a one-way ANOVA.</p

PA700/S10B tyrosine nitration and 26S proteasome sub-complex association (assembly), but not the PA700/S10B protein levels, are increased in aortic homogenates from mouse models of diabetes, hypertension, and dyslipidemia.

<p>Mouse models of (A) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; Tempol, 1 mmol/kg/drinking water, 2 wks.; n = 5/group); (B) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (C) high fat-diets-induced dyslipidemia (LDLr^−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD and for 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed and their homogenates were subjected to immunoprecipitation and Western blot. The immunoprecipitation assay was performed using either an anti-PA700/S10B or anti-3-NT antibody. All blots shown are representative for mice n = 5. All results were analyzed with a one-way ANOVA. * indicates significant <i>vs.</i> control; NS: not significant <i>vs</i>. control.</p

The 26S proteasome is activated and results in degradation of the target proteins, which can be prevented either by ONOO⁻ inhibition or by MG132 administration, in aortic homogenates from mouse models of diabetes, hypertension, and dyslipidemia.

<p>Mouse models of (A) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; MG132, 5 mg/kg/d, i.p., 2d; n = 5/group); (B) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (C) high fat-diets-induced atherosclerosis (LDLr^−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD, 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed and their homogenates were either subjected to 26S proteasome activity assay (chymotrypsin-like activity) (A–C), or Western blotting with the corresponding antibodies as indicated. All results (n = 5) were analyzed with a one-way ANOVA. * indicates significant <i>vs.</i> control; NS: not significant <i>vs</i>. control.</p

Inhibition of the 26S proteasome either by ONOO⁻ inhibition or by MG132 administration rescues endothelial dysfunction in mouse models of diabetes, hypertension, and dyslipidemia.

<p>Mouse models of (A/B) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; Tempol, 1 mmol/kg/drinking water, 2 wks.; n = 5/group); (C/E) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (D/F) high fat-diets-induced dyslipidemia (LDLr^−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD and for 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed for endothelial function assay. The removed aortas were cut into 3-mm rings, and precontracted with 30 nmol/L of U46619 in organ chambers (PowerLab, ADInstruments, Colorado Springs, Co). (A/C/D) Endothelium-dependent vasodilator responses were determined in the presence of acetylcholine (0.01 to 100 µmol/L). (B/E/F) Endothelium-independent vasodilator responses were determined in the presence of sodium nitroprusside (SNP) (0.0001 to 1 µmol/L). All results were analyzed with a one-way ANOVA. * indicates significant <i>v.s.</i> control; NS: not significant <i>v.s.</i> control.</p

ONOO⁻ nitrates PA700/S10B and increases 26S proteasome activity both <i>in vitro</i> and in intact cell.

<p><i>In vitro</i> (A–C): ONOO⁻ (1 µM) was incubated with the purified 26S proteasome for 5 min; in intact cell (D–E): HUVEC was incubated with ONOO⁻ for 0.5 h, in the presence or absence of uric acid (50 µM pre-incubation for 1 h). Cell free system (<i>in vitro</i>) was subjected to (A) Western blot to detect levels of PA700/S10B and the tyrosine nitration of 26S proteasome/PA700/S10B, (B) 26S proteasome activity (chymotrypsin-like activity), (C) an alternative 26S proteasome activity assay: a substrate-in-gel assay with a fluorogenic substrate followed by fluorescence capturing under the UV light. HUVEC cell lysate was subjected to (D) Western blotting of PA700/S10B tyrosine nitration and (E) assay of 26S proteasome activity (chymotrypsin-like activity). All blots shown are representative of three independent experiments. All results (n = 3) were analyzed with a one-way ANOVA.</p

Correction to “Gold Nanoparticles Inhibit Macropinocytosis by Decreasing KRAS Activation”

Correction to “Gold Nanoparticles Inhibit Macropinocytosis by Decreasing KRAS Activation