FIGURE 8.

shRNA knockdown of p42 AUF1 and BLT/c-Raf/ERK1/2 signaling induced a leptomycin B-sensitive nuclear export of p42 AUF1. A, HeLa cells were stably transfected through puromycin selection with the pRS vector or four constructs containing shRNA gene-specific target sequences of AUF1 as described under “Experimental Procedures.” Monolayers were extracted for protein, and 50 μg were analyzed for AUF1 and actin by Western blotting using pan-AUF1 and actin antibodies. B, HeLa cells (3 × 10⁵ in 6-well plates) were transiently co-transfected with 10 ng of luciferase-COX-2-ARE, 500 ng of pcDNA 3, or pcDNA3-p42 AUF1, and 400 ng of pCMV or pRafCAAX as per the illustration. After 16 h, 3 nm leptomycin B or vehicle was added to the wells as indicated for an additional 16 h, after which time monolayers were extracted for nuclear and cytoplasmic protein and 30–50 μg were analyzed for AUF1, GAPDH (cytoplasm), histone H3 (nuclear), and c-Jun (nuclear) by Western blotting. Densitometric analysis in A, Student's t test; pRS versus pRS AUF1-shRNA-4, p < 0.001; pRS versus pRSAUF1-shRNA-1–3, NS, not significant. C, densitometric and statistical analysis of B.