Anti mef2a antibody

According to the needs of different Co-IP experiments, related proteins were overexpressed through gateway system. Briefly, cell lysates were collected using ice-cold IP lysis buffer. The lysates were transferred to a micro centrifuge tube and centrifuged at 13,000×g for 10 min. The supernatant was transferred to a new tube for protein concentration determination and further analysis. The Co-IP analyses were performed using a Co-Immunoprecipitation Kit (ThermoFisher Scientific, #26149) according to the manufacturer's protocol. The experimental steps including: pre-clear lysate using the control agarose resin, Co-IP, elution of Co-IP, resin regeneration and preparation for SDS-PAGE analysis were carried out in turn. Antibodies used therein include: anti-HDAC5 antibody (Invitrogen, PA1-41117, 1:500), anti-Smad7 antibody (Santa Cruz, sc-365846, 1:200), anti-MEF2A antibody (Santa Cruz, sc-17785, 1:200) and anti-NF-κB p65 antibody (Santa Cruz, sc-8008, 1:200). Normal rabbit IgG without antigenicity provided with the kit was used as a negative control. SDS-PAGE and immunoblotting were performed as described above.

ChIP assay was performed using Millipore Chip Kit (#17-10085) according to the manufacturer's protocol. Briefly, cells cultured under the previously indicated conditions were fixed in 1% formaldehyde/PBS for 10 min at room temperature. After two washes with PBS, cells were resuspended in 0.5 mL of lysis buffer containing a protease inhibitor cocktail before sonication. DNA fragments from the soluble chromatin preparations were 400-800 bp in length. Immunoprecipitation was carried out overnight with purified anti-MEF2A antibody (Santa Cruz, sc-17785, 1:200) or anti-NF-κB p65 antibody (Santa Cruz, sc-8008, 1:200) or normal rabbit IgG as a negative control according to a previous study 27 . Protein A/G agarose was used to pull down the antigen-antibody compounds and then washed four times with washing buffers. The DNA-protein crosslinks were reversed with 5 M NaCl at 65 °C for 6 h, and DNA from each sample was purified. PCR was performed using 2 μL DNA samples with the following primers: Smad7: forward 5′-GAATCTTACGGGAAGATCAAC-3′, reverse 5′-CGCAGAGTCGGCTAAGGT-3′.