Fig. 4. Block of ERK5 expression results in the inhibition of myogenic differentiation. (A) ERK5 expression in stably transduced C₂C₁₂ cell lines harbouring empty vector, ERK5WT, ERK5AEF or antisense-ERK5. (B) Cells were seeded at a density of 2 × 10⁵ cells per 10 cm in DMEM/10%FBS and shifted 24 h later to DMEM/2%HS to induce differentiation. Pictures at indicated time points correspond to distinct stages of differentiation. Expression of GFP (last column) is indicative of successfully transduced cells. Bar, 65 µm. (C) Cells were lysed in TLB after 1 and 3 days post-differentiation induction and protein lysates were separated on SDS–PAGE gels. After subsequent immunoblotting, blots were analysed for p21, MyoD and myogenin with the respective antisera described in Methods. Equal protein load of the different samples was controlled with an anti-ERK2 antiserum.