Journal: PLoS ONE

Article Title: Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

doi: 10.1371/journal.pone.0018538

Figure Lengend Snippet: Northern blot analyses of Akap12 expression showing (A) time-dependent increases following atRA stimulation in the indicated cells; (B) retinoid receptor agonist-induced Akap12 mRNA in PAC1 SMC; (C) atRA dose-dependent increase in PAC1 SMC; and (D) RNA polymerase II-dependent increase in PAC1 SMC treated with atRA in absence or presence of actinomycin D (Act-D). Equivalent total RNA loading is indicated with either ethidium bromide staining of 18 S rRNA or expression levels of glyceraldehyde phosphate dehydrogenase ( Gapdh ).

Article Snippet: Primary antibodies used were polyclonal rabbit anti-AKAP12 and mouse anti-PKA RII alpha (BD Transduction Laboratories, Cat # 612242).

Techniques: Northern Blot, Expressing, Staining

Journal: PLoS ONE

Article Title: Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

doi: 10.1371/journal.pone.0018538

Figure Lengend Snippet: (A) Schematic of Akap12 locus comprising three alternate start sites of transcription (bent arrows) under separate promoter control with proper nomenclature for exons. (B) Northern blotting of PAC1 SMC treated with atRA for the indicated times and using exon-specific probes to each Akap12 isoform. Testes RNA (Te) is included as a positive control for the Akap12α and Akap12γ isoforms. (C) Northern blotting with isoform-specific PCR primers on aortic tissue from mice treated with either corn oil or atRA for 24 hr. (D) Western blotting with antisera to AKAP12 on aortic tissue from mice treated with either corn oil or atRA for 24 hr suggests a similar elevation of AKAP12β protein (see also ).

Article Snippet: Primary antibodies used were polyclonal rabbit anti-AKAP12 and mouse anti-PKA RII alpha (BD Transduction Laboratories, Cat # 612242).

Techniques: Northern Blot, Positive Control, Western Blot

Journal: PLoS ONE

Article Title: Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

doi: 10.1371/journal.pone.0018538

Figure Lengend Snippet: Retinoid-induced AKAP12 protein expression.

Article Snippet: Primary antibodies used were polyclonal rabbit anti-AKAP12 and mouse anti-PKA RII alpha (BD Transduction Laboratories, Cat # 612242).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

doi: 10.1371/journal.pone.0018538

Figure Lengend Snippet: (A) Western blot of protein extracts taken from a clone of PAC1 SMC carrying a Myc-tagged AKAP12β transgene stimulated with or without doxycycline (1 µg/ml). Beta actin immunoblot verifies equal protein loading. (B) Parallel dishes of cells carrying Myc-tagged AKAP12β transgene stimulated with or without doxycycline (1 µg/ml) were manually counted with a hemocytometer at the indicated times. Only trypan blue excluding cells were counted. Data are the mean ± SEM of three replicates per time point for each cell line. All three clones carrying AKAP12β showed statistically significant decreases in growth beginning 3 days following Dox stimulation. (C) Western blot showing increases in AKAP12 protein expression within cultured HCASMC transduced with either Ad-AKAP12β (+) or a CMV-driven LacZ adenovirus (−). Blot is representative of two independent experiments. Alpha tubulin immunoblot verifies equal protein loading. (D) Parallel cultures of similarly transduced HCASMC were analyzed for growth over a 5 day period as in panel B. AKAP12β-expressing HCASMC (closed circles) exhibited a statistically significant decrease in growth beginning 3 days following adenoviral transduction as compared to LacZ control cultures (closed squares). Result is representative of two independent experiments performed by different investigators.

Article Snippet: Primary antibodies used were polyclonal rabbit anti-AKAP12 and mouse anti-PKA RII alpha (BD Transduction Laboratories, Cat # 612242).

Techniques: Western Blot, Clone Assay, Expressing, Cell Culture, Transduction

Journal: PLoS ONE

Article Title: Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

doi: 10.1371/journal.pone.0018538

Figure Lengend Snippet: Uninjured right carotid artery (panels A, D) or partial ligation of left carotid artery one week (panels B, E) and three weeks (panels C, F) post-injury were stained for either AKAP12 (red stain in panels A-C) or Ki-67 (brown stain in panels D–F). Arrows point to cells showing clear positivity for Ki-67 and reduced AKAP12. The dotted line in panels C and F represent the full thickness of the neointima. Note the marked decrease in AKAP12 staining after three weeks of the partial ligation injury. Original magnifications were 600×.

Article Snippet: Primary antibodies used were polyclonal rabbit anti-AKAP12 and mouse anti-PKA RII alpha (BD Transduction Laboratories, Cat # 612242).

Techniques: Ligation, Staining

Journal: PLoS ONE

Article Title: Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

doi: 10.1371/journal.pone.0018538

Figure Lengend Snippet: Serial sections of two independent atherosclerotic coronary vessels (panels A–C and D–F) stained for AKAP12 (panels A, D), CNN1 (panels B, E) and Ham56 (panels C, F). The red stain reveals positive immunoreactivity confined largely to the tunica media (AKAP12 and CNN1 indicated with a large arrow) or neointima (Ham56, indicated with small arrow). The neointima is labeled with an asterisk in each panel. Similar results have been found in multiple independent coronary arteries of varying atherosclerotic severity. Magnifications are 200×.

Article Snippet: Primary antibodies used were polyclonal rabbit anti-AKAP12 and mouse anti-PKA RII alpha (BD Transduction Laboratories, Cat # 612242).

Techniques: Staining, Labeling