Bradford protein assay kit

All treated cells were harvested and lysed using RIPA buffer with 0.5 mM PMSF. Cell debris was removed by centrifuge before quantification using Bradford Protein Assay Kit (IBI Scientific). About 20–50 μg of protein was loaded per well on 8–15% SDS-PAGE gel, depending on the expected protein sizes. Proteins in gels were then transferred to polyvinylidene fluoride membranes, which were subject to immunoblotting. Signals were detected using ECL Western Blotting Substrate Kit (Pierce). Images were collected using G:Box XR5 imaging system (Syngene). Primary antibodies: KANK1 (Santa Cruz, sc-135113), CXXC5 (Proteintech, 16513-1-ap), Caspase 3 (Santa Cruz, sc-7148), and ACTB (Santa Cruz, sc-47778). Secondary antibodies: anti-mouse (Proteintech sa00001-1) and anti-rabbit (Proteintech sa00001-2).

HEK293 transfected with single pTAL10-MSTN or co-transfection with MSTN-L and MSTN-R were lysed 48–72 hours post transfection with cold RIPA buffer supplemented with protease inhibitors (1 mM PMSF and 1 mM Benzamidine). Protein concentration was determined by using Bradford Protein Assay Kit (IBI Scientific, Peosta, IA). 20 μg of protein were resolved on a 4-20% Mini-PROTEAN TGX Precast Gel (Bio-Rad Laboratories, Hercules, CA) and transferred onto a PVDF membrane (Millipore, Billerica, MA). Immunoblotting was done with the following antibodies: mouse monoclonal anti-FLAG (F3165; Sigma-Aldrich, Saint Louis, MO), polyclonal anti-HA-Tag antibody (Clontech, Mountain View, CA), mouse monoclonal anti-GAPDH (Millipore, Billerica, MA), and HRP conjugated goat anti-mouse IgG (Millipore, Billerica, MA). Membranes were developed using ECL 2 Western Blotting Substrate (Pierce Biotechnology, Rockford, IL) and images were captured using ChemiDoc XRS + system (Bio-Rad Laboratories, Hercules, CA).