Mef2d

Rabbit polyclonal MEF2A antibody was produced with the assistance of the York University (Toronto, ON, Canada) Animal Care Facility. MEF2D (BD Biosciences, Mississauga, ON, Canada, 610775), KLF6 (Santa Cruz, Dallas, TX, USA, R-173 and E-10), Actin (Santa Cruz, sc-1616), IRDye 680RD goat anti-rabbit (LI-COR, Bioscience, Lincoln, NE, USA) and IRDye 680RD goat anti-mouse (LI-COR, Bioscience) were used for Immunoblotting experiments. FITC- and TRITC-conjugated α-rabbit and α-mouse secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI; D9542), H₂O₂ (H0904) and H89-dihydrochloride hydrate (B1427) were purchased from Sigma Aldrich (Toronto, ON, Canada). Atenolol (Sigma Aldrich, A7655), Isoproterenol hydrochloride (Sigma Aldrich, 1351005) and ICI 118551 hydrochloride (Abcam, Toronto, ON, Canada, ab1200808) were purchased for use in cell culture.

C2C12 mouse myoblasts or human myoblast cell HSMMs were washed with PBS and lysed in RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP 40, 0.1% SDS, 0.5% deoxycholate, Protease Inhibitor Cocktail (Roche)]. Total protein (~20 μg) was separated in SDS-PAGE, transferred to PVDF membranes and immunoblotted with specific antibodies; AKAP6 (1:3000, Covance PRB-451P), AKAP79 (1:3000, BD 610314), MEF2D (1:3000, BD 610774), MyHC (1:3000, Sigma M4276), MEF2C (1:3000, Cell signaling #9792), AKAP-Lbc (1:3000, Santa Cruz sc-9336), AKAP12 (1:3000, Santa Cruz sc-33578), MEF2A (1:3000, Santa Cruz sc-313), myogenin (1:3000, Santa Cruz sc-12732), MyoD (1:3000, Santa Cruz sc-760), actin (1:3000, Santa Cruz sc-1616), α-tubulin (1:5000, Calbiochem CP06). After incubation with horseradish peroxidase conjugated secondary antibody (1:5000, Santa Cruz sc-2005, sc-2004, sc-2020) for 1 h, immunoreactive bands were visualized with enhanced chemiluminescence with Novex® ECL Chemiluminescent Substrate Reagent (Invitrogen).

Antibodies used were against MEF2D (BD Bioscience), H3K27ac (ab4729; Abcam, Cambridge, MA, USA). H3K27me3 (ab195477, Abcam) HDAC4 (Paroni et al., 2004), γH2AX (9718, Cell Signalling, Leiden, The Netherlands), TP53 (DO‐7; Dako, Santa Clara, CA, USA), LT SV40 (sc‐147, Santa Cruz), Lamin B1 (ab16048, Abcam), p21 (CP74, Sigma), RACK1 (sc‐17754, Santa Cruz, Dallas, TX, USA), GFP (Paroni et al., 2004).

The primary antibodies used were as follows: MEF2D (BD Biosciences, San Jose, CA, USA); MEF2A (C‐21 Santa Cruz Biotechnology, Dallas, TX, USA); p53 and pERK (Cell Signaling Technology, Leiden, the Netherlands); p21, Actin, anti‐BrdU, FLAG M2 and RACK‐1 (Sigma‐Aldrich); RAS (Abcam, Cambridge, MA, USA); GFP and HDAC4 (Paroni et al., 2004); and HDAC5 (Clocchiatti et al., 2015). Anti‐CD44‐FITC (BD Biosciences) and CD24‐APC (BioLegend, San Diego, CA, USA) were used with matched control antibodies, anti‐mouse IgG1 FITC and IgG2a APC (Miltenyi Biotec, Bergisch Gladbach, Germany). HDAC7 antibodies were generated in rabbits by injecting recombinant histidine‐tagged HDAC7 fragment aa 261–522. For anti‐HDAC7 antibody purification, HDAC7 was fused to glutathione S‐transferase and cross‐linked to glutathione‐Sepharose as described previously (Paroni et al., 2004).

Cell protein was harvested with ice-cold lysis buffer containing protease inhibitors. Protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then the separated protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA). After 2-h blocking with 5% fat-extracted milk at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against MEF2D (1:3,000; BD) and β-actin (1:4,000; Abcam). Then the membranes were washed with TBST three times and then were treated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized by chemiluminescence detection.