Journal: Epigenetics & Chromatin
Article Title: Ubiquitin ligases HUWE1 and NEDD4 cooperatively control signal-dependent PRC2-Ezh1α/β-mediated adaptive stress response pathway in skeletal muscle cells
Figure Lengend Snippet: Depletion of NEDD4 compromises assembly of PRC2-Ezh1 complex and H3K27me3 occupancy on mCK, MyoG and MYH8 genomic loci under oxidative stress conditions. a , b Interaction between SUZ12 and EED was determined in scramble and NEDD4 knockdown stable cell lines under normal and oxidative stress conditions. Nuclear extracts from scramble and NEDD4 KD cell lines under normal and stress conditions were immunoprecipitated with SUZ12 antibody, associated protein complexes were eluted with 2XLDS loading buffer. SUZ12 and EED were detected using anti-SUZ12 and anti-EED. Protein A Dynabeads alone were incubated with nuclear extracts and used as mock control. Ponceau S staining was used as loading control. c , d ChIP-qPCR analysis of Ezh1α occupancy and H3K27me3 status on genomic loci of mCK enhancer, MyoG promoter, MYH8 and Neurog1. Chromatin immunoprecipitation (ChIP) was performed using chromatin from scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions against Ezh1α or H3K27me3 antibody. Precipitated DNA were measured using qPCR assay with specific primers corresponding to mCK enhancer, MyoG promoter, MYH8 and NeuroG1 genomic regions. ChIP enrichments are shown as percentage of input. Data were expressed as mean ± SD from three biological replicates. Values above each bar indicate Student’s t -test p value. e Transcription level of NEDD4, mCK, MyoG, MYH8 and Atrogin1 were analyzed using RT-qPCR in scramble and NEDD4 KD stable cell lines under normal and oxidative stress conditions. Data were expressed as mean ± SD from three biological replicates. GAPDH was normalized to get relative expression of each target. Values above each bar indicate Student’s t -test p value
Article Snippet: The immunocomplexes were recovered with magnetic Dynabeads (Protein A; Invitrogen) for 2 h and washed on the wheel at 4 °C for 5 min with Low-Salt (LS) wash buffer (0.1% SDS, 2 mM EDTA, 1% Triton X-100, 20 mM Tris–HCl, pH 8, 150 mM NaCl) and High-Salt (HS) wash buffer (0.1% SDS, 2 mM EDTA, 1% Triton X-100, 20 mM Tris–HCl, pH 8, 500 mM NaCl).
Techniques: Stable Transfection, Immunoprecipitation, Incubation, Staining, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing