Figure 5.

Immortalized myogenic cells expressed p19^ARF but sometimes did not express p16^INK4a. Immunoblotting was used to examine p16^INK4a and p19^ARF expression by non-immortalized, rapidly growing, Bax-positive myogenic cells (WT Pass 1); NIH3T3 cells; two independently-derived lines of immortalized Bax-null myogenic cells (BaxKO-1 and BaxKO-2); and one line of immortalized Bax-positive myogenic cells (WT-1). Immunoblotting for Bax was used to verify genotypes, and immunoblotting for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to verify that similar amounts of protein were analyzed in each lane. As expected [14], NIH3T3 cells (immortalized mouse embryo fibroblasts) did not express either p16^INK4a or p19^ARF. Early passage, rapidly growing wild-type cells expressed p19^ARF but not p16^INK4a, though p16^INK4a was expressed when cells entered "crisis" (not shown). The p19^ARF protein was expressed by all immortalized myogenic cell lines that were examined, whereas the p16^INK4a protein was expressed by some (e.g., BaxKO-2), but not all (BaxKO-1) lines. See text for details.