Journal: PLoS ONE

Article Title: Galnt1 Is Required for Normal Heart Valve Development and Cardiac Function

doi: 10.1371/journal.pone.0115861

Figure Lengend Snippet: (A–D) Confocal images and quantification of phospho-histone H3 (pH3) and Ki-67 proliferation markers in developing valves at E11.5 and E12.5. (A) E11.5 OFT cushion tissue (pH3, green; myocardial marker MF20, red; n = 4). (B) E11.5 OFT cushion tissue (Ki-67, green; cell surface marker lectin WGA, red; n = 3). (C) E12.5 OFT cushion tissue (pH3, green; myocardial marker MF20, red; n = 3). (D) E12.5 OFT cushion tissue (Ki-67, green; cell surface marker lectin WGA, red; n = 3). White dash lines in lower magnification in A–D highlight developing cushion area. Higher magnification views are shown to the right of each image. Percentage of nuclei (Nu; blue) positive for pH3 or Ki-67 in wild type samples was set to 1 and relative differences in Galnt1 nulls is graphed to the right of each image. Western blotting reveals an increase in phosphorylation of (E) Smad1/5 (BMP) and (F) MAPK (ERK1/2), (G) decreased EGFR in Galnt1 null OFT samples relative to wild type at E11.5 (n = 4), and E12.5 (n = 6) while remaining unchanged in E10.5 OFT (n = 3) and in E14.5 AoV (n = 3). Samples were normalized to total Smad1, total MAPK and α-Tubulin (α-Tub). (H) Expression of Bmp/Smad genes was examined by qPCR in E12.5 OFT cushion (OFTC) tissues (triplicates, n = 5) isolated by laser-capture microdissection (LCM). While expression of Bmp2 and Bmpr2 remains unchanged, gene expression of Msx1 (a Bmp2 downstream target gene) is significant increased, and Smad6 expression (an inhibitor of BMP signaling) is greatly reduced. Student’s t-test was used to calculate P-valves. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: A, B, C, D = 100 μm for low magnification and 10 μm for higher magnification view.

Article Snippet: For Smad, MAPK and EGFR western blot analyses, membranes were blocked with 5% BSA-TBST and probed with phospho-Smad1/5 and phospho-MAPK (Erk1/2), and total-Smad1 and MAPK antibodies (Cell Signaling #9516P, #4376, #6944, #9102, respectively; rabbit monoclonal for first 3 antibodies and rabbit polyclonal for the fourth antibody; 1:1000), or EGFR antibody (Cell Signaling #4267; rabbit polyclonal antibody; 1:1000) and α-Tubulin antibody (Cell Signaling #2125; rabbit monoclonal antibody; 1:1000).

Techniques: Marker, Western Blot, Phospho-proteomics, Expressing, Isolation, Laser Capture Microdissection, Gene Expression