Mouse monoclonal anti myod

Primary antibodies used in this study are as follows: mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology, sc-32233), mouse monoclonal anti-MYHC (Sigma, 05-716), mouse monoclonal anti-MYOD (Santa Cruz Biotechnology, sc-377460), and mouse monoclonal anti-MYOG (Santa Cruz Biotechnology, sc-12732). Reagents added to the media are as follows: Diethyl succinate (Sigma, 112402), Dimethyl itaconate (Sigma, 592498), Dimethyl malonate (Sigma 136441), Harzianopyridone (Santa Cruz Biotechnology, sc-280769), Itaconic acid (Sigma, I29204), and 4-octyl itaconate (Tocris Bioscience, 6662).

The decapitated embryos and limb bud lysates were prepared, separated by SDS-PAGE, and analyzed by Western blot as described previously (Lewandowski et al. 2017 (link)). The membranes with separated and transferred proteins were incubated with the following primary antibodies: mouse monoclonal anti-Pax3 (Developmental Studies Hybridoma Bank) at dilution of 1:100, mouse monoclonal anti-Lbx2 (Abcam) at dilution of 1:200, rat monoclonal anti-α-actinin (Babraham Bioscience Technologies) at dilution of 1:250, mouse monoclonal anti-MyoD (Santa Cruz Biotechnology) at dilution of 1:200, rabbit polyclonal anti-Myf5 (GeneTex) at dilution of 1:200. Additionally, secondary antibodies were used: donkey anti-mouse IgG HRP conjugated (Jackson ImmunoResearch) at dilution of1:10000, donkey anti-rabbit IgG HRP conjugated (Jackson ImmunoResearch) at dilution of 1:10000, donkey anti-rat HRP conjugated (Jackson ImmunoResearch) at dilution of 1:10000. Membranes were then detected and documented with a chemiluminescent method using the Bio-Rad imaging system. The protein content in the Pax3, Lbx, MyoD, and Myf5 bands was then normalized according to the α-actinin content in each lane.

The primary antibodies used for immunoblotting and immunofluorescence were as follows: rabbit polyclonal anti-IK (Bethyl Laboratories, A301-708A), mouse monoclonal anti-β-actin (Santa Cruz, sc-47778), mouse monoclonal anti-Pax-7 (Santa Cruz, sc-81648), mouse monoclonal anti-MyoD (Santa Cruz, sc-377460), mouse monoclonal anti-myogenin (Santa Cruz, sc-52903), rabbit polyclonal anti-MyHC (Santa Cruz, sc-20641), mouse polyclonal anti-PARP (Cell Signaling, #9542), rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) (Cell Signaling, #9661), rabbit monoclonal anti-Cleaved Caspase-9 (Asp315) (Cell Signaling, #20750), anti-GFP (Cell Signaling, #2956), anti-Bungarotoxin Alexa Fluor™ 647 conjugate (Invitrogen™, #B35450), anti-fast-twitch muscle (DSHB, F310), and anti-slow-twitch muscle (DSHB, F59). The horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Fab) secondary antibodies were purchased from Enzo Life Sciences.