FIG. 3.

C2C12 cells lacking NF-κB activity have an accelerated rate of differentiation. (A) Whole-cell lysates were prepared from C2C12 parental, vector control, or IκBαSR proliferating myoblasts, and 50 μg of sample was used for Western blot analysis. IκBα and IκBαSR proteins were detected with an IκBα polyclonal antibody (C-19; Santa Cruz Biotechnology) at a 1:1,500 dilution. The IκBαSR protein is FLAG tagged and therefore migrates at a slightly higher mobility compared to the endogenous protein. (B) IκBαSR-expressing myoblasts were seeded in triplicate overnight in 12-well plates, and the following day cells were treated with increasing concentrations of TNF-α for 48 h. Cell viability was scored by trypsinization and the trypan blue exclusion method. Cells not treated with TNF-α were designated 100% viable. (C) C2C12 parental cells, vector control, an IκBαSR clone, or five pooled IκBαSR clones were differentiated in DM for 48 h, at which time the cells were prepared for immunofluorescence to detect for the myosin heavy chain. (D) C2C12 vector control or IκBαSR cells were induced to differentiate in DM for up to 72 h. At the indicated times, lysates were prepared and Western blot analysis was performed probing for myogenin expression. V (P) and I (P) denote myogenin expression from five pooled vector control or IκBαSR clones, respectively, that were differentiated for 48 h.