Journal: Circulation Research

Article Title: Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling

doi: 10.1161/circresaha.111.253948

Figure Lengend Snippet: Figure 3. Conditional ILK deletion reduces the activity of the NO/cGMP pathway in intact vessels and endothe- lial cells. A, Immunoblot analysis of total eNOS and ILK in extracts of CT and cKO aortas. Three independent samples are shown (n10 arteries/group, meanSD). B, cGMP production in aortic rings from CT or cKO mice treated with 107 mol/L Ach (NT: untreated, white bars; Ach: black bars) (n6, meanSD; *P0.05 versus NT CT aorta; **P0.05 versus Ach CT aorta). C, Nitrite accumulation in cul- ture supernatants of MAEC isolated from ILK Cre-Lox mice. Cells were treated with vehicle (CT MAEC) or tamoxifen (cKO MAEC) and stimulated with 50 ng/mL VEGF or 106 mol/L A23187 (NT: untreated) (n10, meanSD; *P0.05 versus NT CT MAEC). D, eNOS enzymatic activity in CT and cKO MAEC was mea- sured from the conversion of L-arginine to L-citrulline and superoxide anion produc- tion by flow cytometry detection of DHE (dihydrotehidium) staining (n5, meanSD; *P0.05 versus CT MAEC).

Article Snippet: Circulation Research is available at http://circres.ahajournals.org e48 SUPPLEMENTAL MATERIAL MATERIALS AND METHODS MATERIALS Recombinant human VEGF and ILK antibody were from R&D Systems. eNOS antibody was from BD Biosciences.

Techniques: Activity Assay, Western Blot, Isolation, Cytometry, Staining

Journal: Circulation Research

Article Title: Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling

doi: 10.1161/circresaha.111.253948

Figure Lengend Snippet: Figure 5. ILK deletion leads primarily to eNOS uncoupling. A, Superoxide pro- duction in CT and cKO MAEC treated for 30 minutes with 104 mol/L apocynin, 104 mol/L L-NAME, 104 mol/L allo- purynol, or 105 mol/L rotenone (n5, meanSD; *P0.05 versus CT, **P0.05 versus cKO MAEC). B, Superoxide pro- duction in WT and eNOS KO MAEC trans- fected with ILK-siRNA or nonsilencing ILK-siRNA control (CT siRNA) (see immu- noblot below, n6, meanSD; *P0.05 versus CT siRNA-transfected MAEC). C, Superoxide production in ILK-Cre-Lox MAEC pretreated during 24 hours before tamoxifen deletion of ILK, with either apo- cynin or NAME. Inhibitors were replen- ished daily for 4 days (n6, meanSD; *P0.05 versus nontreated CT; **P0.05 versus nontreated cKO). D, Superoxide production in CT and cKO MAEC non- transfected (white bars) or cKO trans- fected with V5-tagged WT ILK (black bars) (n3, meanSD; *P0.05 versus untransfected CT MAEC; **P0.05 versus nontransfected cKO MAEC). The repre- sentative immunoblot below shows expression of the V5 epitope and overex- pressed ILK (ILK-WT). E, DHFR levels in WT and eNOS KO MAEC transfected with ILK-specific siRNA or nonsilencing siRNA (CT siRNA) (n4, meanSD; *P0.05 ver- sus CT siRNA-transfected WT MAEC).

Article Snippet: Circulation Research is available at http://circres.ahajournals.org e48 SUPPLEMENTAL MATERIAL MATERIALS AND METHODS MATERIALS Recombinant human VEGF and ILK antibody were from R&D Systems. eNOS antibody was from BD Biosciences.

Techniques: Control, Transfection, Western Blot, Expressing

Journal: Circulation Research

Article Title: Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling

doi: 10.1161/circresaha.111.253948

Figure Lengend Snippet: Figure 6. ILK inmunoprecipitates with eNOS. A, Aortic protein lysates from CT and cKO mice were immunoprecipitated with anti-eNOS antibody, and ILK, Hsp90, and eNOS were analyzed by immunoblot. Representative immunoblots for each group are shown (n4, meanSD; *P0.05 versus CT mice). B, Protein lysates from CT and cKO MAEC were immunoprecipitated with anti-eNOS or anti-ILK antibodies, and ILK, Hsp90, and eNOS content was analyzed by immuno- blot (n4, meanSD; *P0.05 versus CT MAEC). C, Protein extracts from human mammary artery (M) or atherosclerotic carotid artery (C) were immunoprecipi- tated with anti-eNOS or anti-ILK antibod- ies, and ILK, eNOS, and Hsp90 levels were analyzed by immunoblot (white bars: mammary artery; black bars: carotid artery; n6, meanSD; *P0.05 versus M).

Article Snippet: Circulation Research is available at http://circres.ahajournals.org e48 SUPPLEMENTAL MATERIAL MATERIALS AND METHODS MATERIALS Recombinant human VEGF and ILK antibody were from R&D Systems. eNOS antibody was from BD Biosciences.

Techniques: Immunoprecipitation, Western Blot

Journal: Circulation Research

Article Title: Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling

doi: 10.1161/circresaha.111.253948

Figure Lengend Snippet: Figure 7. ILK and eNOS interact through Hsp90. A, BAEC were treated with geldanamycin (106 mol/L, 18 hours) and protein lysates were passed through an Ni-NTA agarose column loaded with 6xHis-tagged full-length recombinant eNOS. Bound proteins were eluted and ILK, Hsp90, and eNOS content was ana- lyzed by immunoblot (NT: nontreated; GA: geldanamycin; n3). “Input” shows eNOS, Hsp90, and ILK levels in input samples. Blots are from a representative experiment. B, Protein extracts from MAEC, treated as in A, were immunopre- cipitated with anti-ILK antibody, and ILK, eNOS, and Hsp90 levels were analyzed by immunoblot. A representative experi- ment of 3 is shown C, Effect of ILK over- expression on eNOS complex formation. CT (white bars) and cKO MAEC (gray bars) were untransfected or transfected with V5-tagged WT ILK (black bars) and then immunoprecipitated with anti-ILK. Data are meanSD (n3; *P0.05 versus untransfected CT MAEC; **P0.05 versus untransfected cKO MAEC).

Article Snippet: Circulation Research is available at http://circres.ahajournals.org e48 SUPPLEMENTAL MATERIAL MATERIALS AND METHODS MATERIALS Recombinant human VEGF and ILK antibody were from R&D Systems. eNOS antibody was from BD Biosciences.

Techniques: Recombinant, Western Blot, Over Expression, Transfection, Immunoprecipitation