Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: SMAD3 Deficiency Promotes Inflammatory Aortic Aneurysms in Angiotensin II–Infused Mice Via Activation of iNOS

doi: 10.1161/JAHA.113.000269

Figure Lengend Snippet: Molecular mechanism by which SMAD3 deficiency causes enhanced iNOS‐derived NO production in aorta. A and B, Fluorescence staining for NO, iNOS, and F4/80 in vehicle‐ (A) and AngII‐infused (B) WT and S3KO aortas. Dotted lines demarcate adventitial layer from medial layer. Scale bars=50 μm. C, Western blots showing increased C/EBPβ, phosphorylated NF‐κB, and iNOS in S3KO aorta. Beta‐tubulin served as a loading control. D, In vivo ChIP of C/EBP binding site within the mouse iNOS promoter and a control region 2 kb upstream of the C/EBP site using antibodies against C/EBPβ or preimmune IgG. The gene fragments in the immunoprecipitated chromatin were quantified by RT‐qPCR. Aliquots of the chromatin were also analyzed before immunoprecipitation (input). E, Re‐ChIP was performed using antibodies against coactivator p300. AngII indicates angiotensin II; iNOS indicates inducible nitric oxide synthase; WT, wild type; NO, nitric oxide; S3KO, SMAD3 knockout; C/EBP, CCAAT/enhancer binding protein; L, lumen; m, tunica media; a, tunica adventitia; NF‐κB, nuclear factor‐kappaB.

Article Snippet: Antibodies against CCAAT/enhancer binding protein (C/EBP)–β (#sc‐150), p300 (#sc‐585), α‐smooth muscle actin (α‐SMA [#sc‐69972]), β‐tubulin (#sc‐9104), horseradish peroxidase (HRP)–conjugated goat anti‐mouse IgG (#sc‐2005), and preimmune serum (#sc‐2338) were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Fluorescence, Staining, Western Blot, Control, In Vivo, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Knock-Out

Journal: Journal of cell science

Article Title: Kinase-activity-independent functions of atypical protein kinase C in Drosophila.

doi: 10.1242/jcs.052514

Figure Lengend Snippet: Fig. 5. Biochemical properties of mutant aPKC proteins. (A)Structure of the aPKC protein. The position of the mutations in the four new aPKC alleles is indicated by arrowheads. (B)The GFP-aPKCpsu69 protein does not bind to PAR-6. S2 cells were transfected with wild-type GFP-aPKC and with the four mutant versions of GFP-aPKC. Untransfected S2 cells were used as negative control. The cell lysates were subjected to immunoprecipitation (IP) followed by western blotting (Blot) with the indicated antibodies. Preimmune serum (pre; for PAR-6 IPs) or anti--galactosidase antibodies (-Gal; for GFP IPs) were used as negative controls. (C)The mutant aPKCpsu141 protein is not recognized by the phospho-specific antibody directed against the phosphorylated threonine residue T422. Lysates from S2 cells transfected with GFP-aPKC or with GFPaPKCpsu141 were subjected to western blotting with either an antibody that recognizes aPKC irrespective of its phosphorylation state (aPKC) or with an antibody that specifically recognizes aPKC phosphorylated at T422 (aPKCpT422). A T422 phospho-specific band corresponding to GFPaPKC, but not to GFPaPKCpsu141 was detected. Untransfected S2 cells were used as negative controls. The endogenous aPKC band served as loading control. This observation was confirmed in western blots of adult head extracts of wild-type and aPKCpsu141 homozygous mutant flies (right). (D)Three of the four mutant aPKC proteins show strongly reduced kinase activity. In vitro kinase assays using [-32P]ATP were performed on anti-GFP immunoprecipitates of lysates of S2 cells transfected with either wild-type GFP-aPKC, with any of the four mutant GFP-aPKC versions and with GFP-tagged kinase-dead aPKC (GFP- aPKCK293A). Untransfected cells and cells transfected with wild-type GFP-aPKC but immunoprecipitated with anti-HA antibody were used as negative controls. Both autophosphorylation of GFP-aPKC and phosphorylation of a GST-Baz fusion protein containing the aPKC target site S980 were measured by autoradiography. One representative autoradiogram is shown. The quantitative analysis of four independent in vitro kinase assays is shown in the bar diagrams below the autoradiographs. Values are percentage phosphorylation relative to that of the wild-type GFP-aPKC protein. The input for the kinase assay (top) is shown at the bottom. (E)Three of the four mutant aPKC proteins are kinase-dead with respect to phosphorylation of S980 of Baz. In vitro kinase assays were performed with non-radioactive ATP as phosphate donor and phosphorylation of the GST-Baz fusion protein was detected in western blots using a phospho-specific antibody against S980 of Baz. The immunoprecipitated GFP-aPKC from the same experiment was also detected by western blotting. Quantification of the blots with the phospho-specific Baz antibody from four independent experiments and the GST-Baz input corresponding to the kinase assay (top) are shown at the bottom.

Article Snippet: For immunoprecipitations, 2 l of rabbit anti-GFP (Molecular Probes #A11122), 2 l of guinea pig anti-PAR-6, 2 l of the corresponding preimmune serum, 10 l of rabbit anti--galactosidase (MP Biomedicals #55976) or 2 l of rat anti-HA 3F10 (Roche) were added to cell lysate containing 500 g of total protein from S2 cells in TNT (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl pH 7.5) supplemented with protease inhibitors.

Techniques: Mutagenesis, Transfection, Negative Control, Immunoprecipitation, Western Blot, Residue, Phospho-proteomics, Control, Activity Assay, In Vitro, Autoradiography, Kinase Assay