C2C12 Cell Treatment with Pharmacological Agents

C2C12 cells were treated with different drugs: TubA (at 5 µM, APExBIO, #A4101), tubacin (TBC, 5 µM, Sigma, #SML0065), rapamycin (rapa,100 nM, Sigma #CAS 53123-88-9) and SB 431542 (SB43, 10 µM, Tocris, #1614). Recombinant Human TGF-β1 (rhTGF-β1, 10 ng/mL, #100-21) was purchased from PeproTech. All primary antibodies used in this study are presented in supplementary table 1. Secondary antibodies used for immunofluorescence studies were coupled to Alexa-Fluor 488 or Alexa-Fluor 555 (Molecular Probes); or to Cy3 or Cy5 (Jackson ImmunoResearch Laboratories). Secondary antibodies used for Western blotting were either horseradish peroxidase (HRP)-coupled anti-rabbit-IgG polyclonal antibodies (Bio-Rad) or HRP goat anti-mouse-IgG antibodies (Bio-Rad). All antibodies used in this study were validated by the manufacturers or in previously published work from the lab. To visualize NMJ for immunofluorescence studies, we used α-Bungarotoxin at 5 µg/mL conjugates with Alexa-Fluor 488 (Molecular Probes, # B13422) and DAPI (Sigma-Aldrich; # D9542) was used to stain nuclear DNA. To visualize and quantify proteins on Western blot, we used 2,2,2-Trichloroethanol^{122 (link)} (TCE, Sigma, #T54801).