Fig. 2. Notch activity is essential for the maintenance of Pax7-positive cells during myogenic differentiation in vitro. (A) Primary myoblasts, incubated in growth medium until 90-100% confluent, were transferred to differentiation medium and were incubated for 1 day in the presence of DMSO or 5 µM GM6001, a metalloproteinase inhibitor (left), or in the presence of DMSO or 1 µM DAPT, a -secretase inhibitor (right). The levels of NICD, Pax7, Pax3, MyoD, myogenin and p21 were determined by western blotting, tubulin is a gel-loading control. A representative experiment out of three is shown. (B) Primary myoblasts incubated for 1 day in differentiation medium in the presence of DMSO, 5 µM GM6001 or 1 µM DAPT were stained with mouse anti-Pax7 and goat anti-desmin antibodies, and then with Rhodamine-Red-X-conjugated anti-mouse IgG and AMCA-conjugated anti-goat IgG antibodies. (C) Confluent C2C12 cells were incubated for 1 day in differentiation medium in the presence of DMSO or 5 µM DAPT and the levels of NICD, Pax7, Pax3, MyoD, myogenin, p21 and tubulin were analyzed by western blotting.