Figure 5.

RHA down-regulation reduces JUND polysomes. Cyto lysates of HEK293 cells transfected with siRNA targeting NCBP3 (siNCBP3), RHA (siRHA), or nontargeting siRNA (siNT) were subjected to WB and RT-qPCR. A, representative immunoblot of the cyto lysates with antiserum against RHA, NCBP3, NCBP1, or the loading control GAPDH. The antiserum detected the specific proteins on the immunoblots, as shown relative to the prestained molecular mass markers (M). The same image of the molecular mass markers was used for each panel. B, dhx9/rha and ncbp3 expression in cells treated with the indicated siRNA by RT-qPCR. The bar graph represents the expression of dhx9/rha and ncbp3 relative to GAPDH. C, A₂₅₄ spectrometry of sucrose gradient showing rRNA distribution and designation of the fractions. PIC, preinitiation complex composed of 40S and 60S RNPs; Mono, monosome (80S); LP, light polysome (two or three polysomes); HP, heavy polysome (4 or more polysomes). D, RT-qPCR of RNA extracted from the fractions identified in C for expression of JUND and GAPDH. Copies were calculated relative to standard curves. The graphs present the distribution of the RNA copies across the gradients. The results represent the means of three independent experiments (bars) with standard deviation. The colored circles indicate the values from the individual experiments. Statistical significance is indicated with asterisks: *, p ≤ 0.05; **, p ≤ 0.005.