Fig. 4. Upregulation of INK4b in senescent CEFs. (A) Growth curve of primary CEFs passaged continuously until they reached senescence. (B) Photomicrographs of young and senescent CEFs after staining for senescence-associated (SA)-ß-galactosidase activity. (C) Northern blot of RNA from CEFs at 27, 43 and 53 PDs, hybridised with probes for the unique 3'-untranslated regions of INK4b and ARF, or with a genomic DNA fragment that recognises both transcripts [described as probe 1 in Kim et al. (Kim et al., 2003)]. The p21^CIP1 RNA was detected using a chick p21^CIP1 cDNA probe and GAPDH served as a control for loading. (D) Equivalent samples of total protein from CEFs at 27, 43 and 53 PDs were analysed by immunoblotting for endogenous INK4b (SK14 antibody) and p53. Actin served as loading control.