FIG. 2.
Kinase activity in senescent-cell nuclear extracts inhibits SRF DNA binding activity. (A) EMSAs with 32P-labeled SRE oligonucleotide and SRF(His)6 previously incubated with nuclear extracts from young (Y) or old (O) nuclear extracts are shown in lanes 1 and 2. Parallel kinase reactions were incubated with mutant (mut) SRE (lanes 3 and 4), competed with a 100-fold excess of unlabeled wild-type (WT) SRE (lanes 5 and 6), or incubated with a 100-fold excess of unlabeled mutant SRE (lanes 7 and 8). Preincubation with polyclonal SRF antibody (αSRF, lanes 9 and 10) supershifted or eliminated the SRF complex. Parallel reactions without SRF(His)6 did not form complexes. (B) SRF(His)6 was used in kinase reactions with equal amounts of young (Y), senescent (O), or a combination of young- and old-cell nuclear extracts (Y/O) and used in SRE EMSAs (lanes 2 to 4). A control (C) kinase reaction with SRF but without nuclear extract is shown in lane 1. Parallel reactions were also done in the presence of the phosphatase inhibitors sodium fluoride (NaF) and sodium vanadate (Na-Van) (lanes 5 to 7). The proportional addition of senescent (Old%) nuclear extracts to young nuclear extracts (Young%) were also used in SRE EMSAs (lanes 8 to 12). (C) Reactions with SRF(His)6 incubated with kinases supplied from equal amounts of young (Y), senescent (O), or a combinations of young and old nuclear extracts (Y/O) with 10 μM ATP were used in SRE EMSAs (lanes 2 to 4). A control (C) reaction with SRF but without nuclear extract is shown in lane 1. Parallel reactions were also carried out in the absence of ATP used in EMSAs (lanes 5 to 8). (D) Data were obtained from SRE EMSAs utilizing SRF kinase reactions in the presence of SRF(His)6 as the substrate and kinases supplied from equal amounts of young (Y) and senescent (O) nuclear extracts. Various amounts of the PKC inhibitors bisinodolylmaleimide II (Bis II; 25, 50, and 100 nM), chelerythrine chloride (CH-Cl; 1.25, 2.5 and 5 μM), rottlerin (Rot; 5, 10, and 20 μM), or dimethyl sulfoxide (DMSO, 1%) vehicle were used in each reaction. Control reactions with SRF but in the absence of nuclear extracts were also followed by EMSA and used to normalize experiments. Histograms show data from scanning densitometry of three independent EMSAs with the average ratio of young and old intensities relative to the control reaction under each drug concentration, with standard deviations shown by error bars.