Fig. 1. Notch activity in myogenic cells during differentiation in vitro. Primary mouse myoblasts or C2C12 cells were incubated in growth medium until they were 90-100% confluent (GM, Day 0) and then they were transferred to differentiation medium (DM, Day 1 to Day 3). (A) The levels of active Notch-1 (NICD), Pax7, MyoD, myogenin, p21 and tubulin were determined by western blotting. NICD was detected using epitope-specific antibody against -secretase-cleaved Notch-1. (B) The amount of NICD in A was quantified by gel densitometry and normalized to the amount of tubulin (black bars); the amount of NICD at Day 0 is set as 1. Percentage of BrdU-positive cells was determined after 3-hour-pulse BrdU labeling (gray bars). Experiments in A and B were repeated three times with similar results; representative experiments for primary myoblasts and C2C12 are shown. (C) C2C12 cells incubated for 3 days in differentiation medium were subjected to partial trypsinization to separate myotubes (Mt, integrin 7A-positive) from reserve cells (RC, Pax7-positive). The amount of NICD in Mt and RC fractions and in total cell lysate was determined by western blotting.