Figure 6

PED/PEA-15 autophagy-promoting activity mediates TGF-beta1 action on L6 cell differentiation. L6 cells stably overexpressing PED/PEA-15 (pcDNAIII-PED) or transfected with the empty vector (pcDNAIII) were plated and enabled to differentiate as described under Materials and Methods. AVO accumulation was determined as outlined in Figure 4 (a). The one shown is representative of four independent experiments. (b) Alternatively, the cells were lysed and 100 microgram protein samples were analyzed by western blotting using the indicated antibodies. Blots were revealed by ECL and autoradiography, using beta-actin as loading control. The autoradiograph shown is representative of three independent experiments. (c) Differentiated myotubes were transiently transfected with either the ShPED/PEA-15 RNA (Sh PED/PEA-15) or a scrambled RNA (Sc) and then exposed to 5 ng/ml TGF-beta1. AVO accumulation was assessed as outlined in Figure 4. The one shown is representative of three independent experiments. (d) Alternatively, the cells were lysed and protein samples were analyzed by western blotting using the indicated antibodies. Blots were revealed by ECL and autoradiography, using beta-actin as loading control. The autoradiograph shown is representative of four independent experiments