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3 protocols using anti mef2a

1

Immunofluorescence Staining of Leukemia Cells

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C8166 and MT-2 cells were cultured overnight on glass coverslips coated with poly-L-lysine. Cells were fixed with 1% paraformaldehyde and permeabilized with 0.2% Triton X-100. Fixed cells were incubated with SuperBlock buffer (Thermo Scientific) followed by staining with a Tax hybridoma (AIDS Research and Reference Program), and either anti-MEF-2A (Santa Cruz), anti-CREB (48H2; Cell Signaling), or anti-IRAK1 (D51G7; Cell Signaling) rabbit antibodies. Cover slips were incubated with Alexa Fluor 555-conjugated donkey anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Life Technologies) and DAPI. Images were obtained using a Nikon C1si confocal microscope.
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2

Immunofluorescence Imaging of Myogenic Markers

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C2C12 mouse myoblasts or human myoblast cell HSMMs were seeded on μ-Dish35 mm high (ibidi), allowed to grow up to 70–80% cell confluence, and switched to differentiation media. After differentiation, cells were fixed with 4% PFA at 4 °C for 10 min, blocked with 2% BSA-PBS solution for 1 h, and labeled with anti-AKAP6 (1:400, Covance PRB-451P), anti-myogenin (1:400, Santa Cruz sc-12732), anti-MyHC (1:400, Sigma M4276), or anti-MEF2A (1:400, Santa Cruz sc-313) overnight at 4 °C, followed by fluorescent dye conjugated secondary antibody (1:200, Invitrogen A-21202, A-31572). The nuclei were stained with DAPI (Molecular probe) and mounted using fluorescent mounting medium (DAKO). The fluorescent images were obtained using a confocal microscope (Carl Zeiss LSM710).
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3

ChIP-exo Profiling of MEF2A Transcription Factor

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Note that 15 × 106 C2C12 (48 h DM) and 8 × 106 primary rat CMs were prepared for ChIP-exo as follows: Cells were washed with 1× PBS and treated with 37% formaldehyde (Sigma-Aldrich) for 15 min at 37°C. The cell pellet was isolated similar to ChIP-qPCR as previously described (35 (link)). DNA was sonicated to ∼250 bp in length. Cross-linked chromatin was sent to Peconic Genomics with 5 μg anti-MEF2A (Santa Cruz) and Rabbit IgG (Millipore). Peconic Genomics completed ChIP-exo as previously described (31 (link)) and the resulting samples were sequenced on the Illumina HiSeq 2000 platform. Illumina CASAVA software was used for base calling and sequencing reads were aligned to the mm10 (MBs) or rn5 (CMs) genome assembly using BWA 0.5.9 (36 (link)). Raw data were filtered for a quality score of 37 using SAMtools (37 (link)), and duplicates were removed using Picard (http://picard.sourceforge.net/). MACS 1.4.2 was used to do peak calling analysis (38 (link)). To identify MEF2A target genes in skeletal and cardiac muscle corresponding to peak location, MEF2A enrichment peaks identified in MACS were converted to mm9 using UCSC LiftOver (39 (link)).
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