FIG. 4.
Transcriptional activation of p21 and mdm2 genes by p53 is enhanced by PKR. (A and B) PKR+/+ and PKR−/− cells expressing ts p53 were either maintained at 37°C (lanes 1 and 6) or switched to 32°C for 1 (lanes 2 and 7), 3 (lanes 3 and 8), 6 (lanes 4 and 9), and 12 (lanes 5 and 10) h. (A) Northern blot analysis. Total RNA (10 μg) was subjected to Northern blot analysis using a mix of 32P-labeled mdm2 and mouse p21 cDNAs as a probe (top panel). The levels of mdm2 and p21 RNA expression were normalized to actin RNA (bottom panel). (B) Immunodetection of p21 levels in PKR+/+ and PKR−/− cells expressing ts p53. Cell extracts (50 μg of protein) were subjected to SDS-PAGE (12% gel), electrotransferred to a PVDF membrane, and probed with an anti-p21 antibody. The blot was stripped and reprobed with an antiactin antibody for normalization of protein extracts. (A and B) Quantification of bands was performed by scanning autoradiograms in the linear range of exposure with a Chemiimager 4000 imaging system and analyzing with spot densitometry software (Alpha Innotech Corporation).