Figure 5.

Glycerol gradient centrifugation resolves the RNA-stimulated kinase activity from the basal C1 hnRNP protein phosphorylation activity. Micrococcal nuclease-treated HeLa nuclear extract was layered on 10–30% (vol/vol) glycerol gradients and centrifuged for 18 hr at 40,000 rpm (Beckman SW41 rotor) at 4°C. The gradients were fractionated, and each fraction was divided into two equal portions. One was assayed for C1 hnRNP protein phosphorylation without exogenous RNA, and the other was assayed for C1 hnRNP protein phosphorylation in the presence of added nuclear RNA (final concentration, 125 μg/ml). The proteins were displayed by electrophoresis and autoradiograms exposed in the linear range of the film’s dpm vs. silver grain exposure curve were subjected to quantitative densitometry. The amounts of ³²P in the C1 hnRNP protein bands were summed and plotted as a function of gradient position. •, No added RNA; ○, with nuclear RNA. The S values indicated by the arrows were estimated by interpolation of the gradient positions of apoferritin (18S) and alcohol dehydrogenase (8S) standards (18).