Journal: PLoS ONE
Article Title: Smooth Muscle Cell Genome Browser: Enabling the Identification of Novel Serum Response Factor Target Genes
Figure Lengend Snippet: Identification of an SMC-specific Ryr3 regulated by SRF. (A) A genomic map view of Ryr3 variants expressed in JSMCs and CSMCs. Three variants TCONS_00167498 (V498, blue), TCONS_00152671 (V671, green), and TCONS_00166589 (V589, red) are indicated by arrows. (B) Expression levels of total Ryr3 in SMCs. (C) Expression levels of each Ryr3 vaiant in SMCs. Variants are arranged on the X-axis from longest (left) to shortest (right) in length. The three variants shown on A are also indicated by the color arrows. (D) A topological map of RYR3 variants. Each circle denotes the corresponding amino acid. Colors on amino acid sequence show distinct regions and domains: red, missing or inserted peptides from differentially spliced exons; green, start codons found in differentially spliced variants. Seven transmembrane domains 1–7 and a pore region (magenta) are shown. MR1-5 (blue), SPRY1-3 (dark green), FKBP1A (orange), CALM (purple), and a residue E (black) that is required for Ca 2+ binding (Ca 2+ ) are also indicated. The topology map was drawn based on the longest peptide variant V498. V671 is a C-terminal truncated variant (V671CT) that is missing Ca 2+ binding and transmembrane domains after the CALM domain while V589 is an N-terminal truncated variant (V589NT) that is missing the N-terminal domains (N-ter to CALM domain). (E) Western blot showing that RYR3 protein expression decreased in jejunum SM of inducible SMC-specific Srf KO mice as SRF protein was depleted 5, 10, 15, and 20 days following tamoxifen administration. UBE1 was used as an endogenous control.
Article Snippet: Primary antibodies against the following antigens were used: RYR3 (rabbit, 1:1200, Alomone Labs, Jerusalem, Israel), SRF (rabbit, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), or GAPDH (mouse, 1:1500, Abcam, Cambridge, MA, USA).
Techniques: Expressing, Sequencing, Binding Assay, Variant Assay, Western Blot, Mouse Assay