FIG. 2.

Myogenic differentiation correlates with loss of NF-κB transactivation function. (A) C2C12 myoblasts stably containing a 3xκB-Luc reporter plasmid were propagated as either a mixed population or a clonal isolate. Cells were plated in triplicate overnight in 6-cm culture dishes, and on the following day they were maintained in GM or switched to DM for 48 h. At that time, cell extracts were prepared, and relative luciferase units were determined by normalizing to total protein (RLU). Promoter activities were also determined for 3xκB-Luc and 3xκBmut-Luc populations that were treated or not treated with 10 ng of TNF-α per ml for 24 h. (B) C2C12 cells were maintained in GM or differentiated in DM for up to 72 h. At the indicated times, total RNA was prepared and 10 μg of sample was used for Northern blot analysis. The blot was hybridized with an IκBα-specific probe, and RNA loading was normalized by stripping the blot and reprobing for GAPDH mRNA.