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1.
Fig. 3.

Fig. 3. From: Myocardin inhibits cellular proliferation by inhibiting NF-?B(p65)-dependent cell cycle progression.

p65 inhibits the formation of myocardin/SRF/CArG ternary complex. (a) Myc-tagged myocardin, Flag-tagged p65, and SRF proteins were expressed by in vitro transcription and translation and incubated with radiolabeled probes, and gel mobility shift assays were performed. (b) Gel mobility shift assays were performed with increasing amounts of p65 (Left) and myocardin (Right). (c) Gel mobility shift assays were conducted from mouse hearts with or without LPS. Anti-SRF antibodies were applied for supershift. p65 protein was analyzed by Western blot.

Ru-hang Tang, et al. Proc Natl Acad Sci U S A. 2008 March 4;105(9):3362-3367.
2.
Fig. 5.

Fig. 5. From: Myocardin inhibits cellular proliferation by inhibiting NF-?B(p65)-dependent cell cycle progression.

Myocardin inhibits CHO cell proliferation. (a) Numbers of cells harboring the myocardin-inducible system were counted 72 h after treatment with or without Dox (n = 7). *, P < 0.01. (b) Cells were treated for 3 days, pulse-labeled with BrdU for 60 min, and stained with BrdU (for both cell count and BrdU experiments (n = 7). *, P < 0.01. (c) Representative DNA histograms obtained through laser scanning cytometry in M33 CHO cells treated for 24 h. (d) Cell numbers in both G2–M phase and polyploid cell regions as detected by cytometry (n = 5). *, P < 0.01. (e) Western blot with corresponding antibodies for cell-cycle regulatory proteins in cells with (+) and without (−) Dox treatment for 3 days.

Ru-hang Tang, et al. Proc Natl Acad Sci U S A. 2008 March 4;105(9):3362-3367.
3.
Fig. 2.

Fig. 2. From: Myocardin inhibits cellular proliferation by inhibiting NF-?B(p65)-dependent cell cycle progression.

Physical interaction between p65 and myocardin. (a) COS7 cells transfected with expression plasmids encoding full-length myocardin fused to GAL4 and UAS-luciferase reporter, with or without expression plasmid for p65. Values are the fold increase of luciferase activity relative to activation of the reporter alone in at least three experiments. Student's t test: P < 0.05, Gal4-myocardin vs. Gal4-myocardin plus p65. (b) Anti-Myc antibodies detected myocardin in immunoprecipitates prepared with anti-Flag antibodies and COS7 cells transfected with Flag-p65 and Myc-myocardin expression plasmids. (c) (Left) Coomassie-stained proteins corresponding to the amounts of GST and GST-myocardin protein in the pull-down assay are shown. (Right) Summary of p65 interaction with myocardin domains. (d) Autoradiogram indicating 1/20 input of radiolabeled p65 proteins used in pull-down assay. (e) Interaction between full-length and truncated p65 and GST-myocardin (Left) and GST alone (Right). (f) Summary of myocardin interaction with p65 domains.

Ru-hang Tang, et al. Proc Natl Acad Sci U S A. 2008 March 4;105(9):3362-3367.
4.
Fig. 1.

Fig. 1. From: Myocardin inhibits cellular proliferation by inhibiting NF-?B(p65)-dependent cell cycle progression.

Suppression of myocardin transcriptional activity by p65. Luciferase reporters controlled by SM22 and ANF were transfected into COS7 cells (a) or rat neonatal cardiomyocytes (b) and cotransfected with expression plasmids for myocardin (0.1 μg) and increasing amounts of p65 (COS7: 0.01, 0.05, 0.1, and 0.2 μg; cardiomyocytes: 0.02 and 0.1 μg). p65 protein assayed by Western blot. (c) COS7 cells were cotransfected with the indicated deleted CArG box within either the SM22 or ANF promoter. (d and e) COS7 cells (d) or cardiomyocytes (e) treated with TNF-α (10 ng/ml for 6 h) or cotransfected with an IκBα superrepressor. Values are the fold increase of luciferase activity relative to activation of the reporter alone of at least three experiments. Student's t test: P < 0.05, myocardin alone vs. myocardin plus p65, TNF-α, or IκBα-SR.

Ru-hang Tang, et al. Proc Natl Acad Sci U S A. 2008 March 4;105(9):3362-3367.
5.
Fig. 6.

Fig. 6. From: Myocardin inhibits cellular proliferation by inhibiting NF-?B(p65)-dependent cell cycle progression.

Effect of endogenous myocardin on VSMC growth. (a) Photomicrographs of VSMC harboring the tetracycline-regulated (TR) inducible system for myocardin and its dominant negative treated with and without Dox for 24 h. (b) Cell numbers counted over 3 days in control (TR), myocardin (Myocd), and dominant negative (Myocd-DN) expressing VSMC (n = 5). *, P < 0.01. (c) Mouse aortic VSMC were transfected with siRNA to either myocardin or p65 for 24 h. Real-time PCR was performed to detect relative fold changes in mRNA expression (normalized to 18s rRNA). Values are the relative fold change in mRNA expression relative to control of at least three experiments. P < 0.05 for both siRNA vs. control and siRNA myocardin vs. siRNA p65. (d) VSMC directly counted 48 h after being transfected with either myocardin or p65 siRNA. Error bars represent SD of five experiments. P < 0.05 for both siRNA vs. control and siRNA myocardin vs. siRNA p65. (e) Working model balancing NF-κB(p65) and myocardin regulation of cellular growth and differentiation.

Ru-hang Tang, et al. Proc Natl Acad Sci U S A. 2008 March 4;105(9):3362-3367.
6.
Fig. 4.

Fig. 4. From: Myocardin inhibits cellular proliferation by inhibiting NF-?B(p65)-dependent cell cycle progression.

Myocardin repression of p65 transcriptional activity. (a) COS7 cells transfected with a luciferase reporter construct of three tandem κB sites or its mutant, together with expression plasmids for p65 (0.1 μg), myocardin (0.1 and 0.2 μg), or both. Values are the fold increase in luciferase activity relative to activation of the reporter alone. Error bars represent SD of at least three experiments. Student's t test: P < 0.05, p65 alone vs. p65 plus myocardin. (b) Gel mobility shift assays were performed with a 32P-labeled κB probe with stable levels of in vitro translated p65 with or without increasing amounts of in vitro translated myocardin. (c) Aortic VSMCs infected with increasing amounts of adenoviral myocardin (multiplicity of infection: 20, 50, and 200) for 48 h and then stimulated with LPS (1 μg/ml) for 2 h. Western blots were performed on extracted proteins.

Ru-hang Tang, et al. Proc Natl Acad Sci U S A. 2008 March 4;105(9):3362-3367.

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