Cy3 or cy5

OBSCs were fixed for 20 min in 4% PFA in phosphate buffered saline (PBS), washed three times in PBS, then blocked for 1 h in PBS supplemented with 0.5% Triton X-100 and 3% normal Goat Serum (Sigma G9023). Slices were incubated at 4 °C in primary antibody (diluted in blocking solution) overnight. In order to detect PDGFRβ, heat-mediated antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) for 40 min at 80 °C prior to primary antibody incubation. Slices were washed a further three times in 0.5% Triton-X100 in PBS (PBS-TX) then incubated with secondary antibodies (Life Technologies and Jackson) (1:500 dilution in blocking solution for 2 h at 4 °C). Three final PBS-TX washes were conducted before slices were mounted on slides and images captured using a Leica Confocal Microscope. Primary antibodies used: rabbit anti-PDGFRβ (28E1) (1:200, Cell Signalling, Cat. No: 3169S), rat anti-PECAM-1 (1:400, BD, Cat. No: 550274), rabbit anti-laminin (1:200, Sigma, Cat. No: L9393), Mouse MOAB-2 (pan Aβ) (1:1000, Merck-Millipore, Cat. No: MABN254) Rabbit Ki67 (1:1000, Abcam, Cat. No: ab15580) secondary staining was conducted using species-specific fluorophore-conjugated (Streptavidin Alexa 488, Molecular Probes; Cy3 or Cy5, Jackson,) or biotin-conjugated secondary antibodies (Jackson). DAPI (1 μg/mL, Sigma) was used to counterstain nuclei.

All primary antibodies used in this study are presented in Table 1. Secondary antibodies used for immunofluorescence studies were coupled to Alexa-Fluor 488 or Alexa-Fluor 546 (Molecular Probes) or to Cy3 or Cy5 (Jackson ImmunoResearch Laboratories). Secondary antibodies used for Western blotting were either HRP coupled anti–rabbit-IgG polyclonal antibodies (Jackson ImmunoResearch Laboratories) or HRP goat anti–mouse-IgG antibodies (Millipore). To visualize the NMJ for immunofluorescence studies, we used α-BTX at 5 µg/ml conjugates with either Alexa-Fluor 488 or Alexa-Fluor 594. All drugs used in this study are presented in Table 2.

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).