Construction and purification of nanoswitches
Cell culture and differentiation assay
Total RNA extraction and small RNA isolation
Immunocytochemistry
qRT-PCR for miRNAs
miRNA detection
Gel imaging and analysis
Cells harvested either from GM or from different stages of DM were lysed in lysis buffer containing 50 mM tris-HCl, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, and 10% of glycerol supplemented with protease inhibitor mix (Sigma-Aldrich). Cell lysates were centrifuged at 10,000 rpm for 5 min at 4°C to get rid of cell debris. Protein concentration was estimated by bicinchoninic acid assay (Pierce) using a plate reader. Proteins were separated in SDS–polyacrylamide gel electrophoresis, transferred, and immunoblotted with various antibodies. The antibodies used were mouse monoclonal antibody, anti-MHC, anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Sigma-Aldrich), and anti-Myog (Santa Cruz Biotechnology).