Anti myf5 c 20

Chromatin immunoprecipitations were performed on Rh18 cells using the Chip-IT High sensitivity kit (Active Motif, Carlsbad, CA) and anti-H3K27ac (Active Motif) or anti-MYF5 (C-20, Santa Cruz) antibodies. Resultant purified immune-precipitated DNA was used for library preparation using the TruSeq ChIP sample preparation kit (Illumina, San Diego ,CA) without modifications. 11–18 library preps were mixed for multiplexed single read sequencing using the NextSeq500 (Illumina).Previously published MYOD ChIP-seq data from RD was downloaded and processed in parallel with the newly generated sequencing data (GSE50415, GSE84630) (MacQuarrie et al., 2013a (link)). Reads were aligned to the hg19 reference using BWA. ChIP-seq peaks were identified using MACS 2.1 (Zhang et al., 2008 (link)). Gene ontology was performed using GREAT (McLean et al., 2010 (link)). Differential peak calling between RD and Rh18 was performed using bedtools v2.25.0 and visualized using NGS plot (Shen et al., 2014 (link)). Genomic regions were visualized using IGV v2.3.40.

Immunodetection was performed on X-Gal stained 20 µm cryostat sections, using monoclonal mouse anti-Pax3-C antibodies (DSHB) and the Vector M.O.M (Mouse on Mouse) Peroxidase kit (Vector Laboratories). Sections of control and Msx mutants shown in Fig. 5A were 10 µm thick. Monoclonal mouse anti-Pax3-C antibodies (1:250, DSHB), rabbit polyclonal anti-Myf5-C20 (1:250, Santa Cruz), Alexa Fluor 488 Anti-Mouse IgGs and Alexa Fluor 546 Anti-Rabbit IgGs (1:500, Molecular Probes) were employed for immunofluorescence experiments. Nuclear staining was with Hoechst solution.

For Whole Cell Extracts preparation, cells were pelleted by centrifugation, resuspended in ice-cold RIPA buffer (10 mM TrisHCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.1% sodium deoxycholate, 140 mM NaCl, 1% Triton X-100, 1 mM PMSF, Protease inhibitor cocktail) and incubated for 30 min on ice, with occasional shaking. Samples were centrifuged at 13,000 rpm for 10 min at 4°C and the supernatant recovered and quantified using the Bradford protein assay.
20 μg of extracts were loaded on a 4–10% SDS-polyacrylamide gel and analyzed by Western blot using primary antibodies and a peroxidase-conjugate secondary antibody (Sigma-Aldrich). Primary antibodies: anti-NF-YA (G-2, Santa Cruz), anti-NF-YB (GeneSpin), anti-NF-YC (home-made) anti-Vinculin (H-10, Santa Cruz), anti-MyHCs (MF20, DHSB), anti-Myogenin (IF5D, DHSB), anti-MyoD (C-20, Santa Cruz), anti-Myf5 (C-20, Santa Cruz), anti-Pax3 (DHSB), anti-Snai1 (C15D3, Cell Signaling). Western blot experiments were performed on three independent biological replicates.