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. 2009 Mar 16;29(10):2622–2635. doi: 10.1128/MCB.01495-08

FIG. 8.

FIG. 8.

(A) Half-lives of HO-1 and GAPDH mRNAs were calculated for IMR-90 cells that were transfected with either control siRNA or HuR siRNA by using Act D as described in the legend to Fig. 3A. (B and C) IMR-90 cells were transfected with either control siRNA or HuR siRNA, together with either a control plasmid (pTAP) or a plasmid expressing the HuR-TAP chimeric protein; 48 h later, cells were left untreated or were treated with NO (0.5 mM for 4 h). The levels of HO-1 mRNA and those of normalization control 18S rRNA were measured by RT-qPCR (B), and the levels of HO-1 protein, endogenous and TAP-tagged HuR, and loading control β-actin were assessed by Western blot analysis (C). (D) Nascent HO-1 production was monitored following a brief (20-min-long) incubation of IMR-90 cells with l-[35S]methionine and l-[35S]cysteine after either no treatment (−) or treatment with NO (0.5 mM for 1 h). Following IP using either anti-HO-1 antibody, anti-GAPDH antibody, or control IgG, the incorporation of radiolabeled amino acids into the newly synthesized HO-1 and GAPDH proteins was assessed by electrophoresis through 12% SDS-containing polyacrylamide gels and visualization and quantitation using a PhosphorImager apparatus. (E) The relative association of HO-1 and GAPDH mRNAs with polysomes was tested by preparing cytoplasmic lysates from cells treated as described above (D), fractionating them through sucrose gradients, and collecting 10 fractions for analysis. RNA was extracted from each fraction, and the levels of HO-1 mRNA in each fraction from each population (untreated, top left graph [Untr.]; NO treated, bottom left graph) were measured by RT-qPCR. The levels of GAPDH mRNA were also measured in each fraction and plotted (right graphs). The data shown are representative of three independent experiments. The direction of sedimentation (arrows) as well as the components of the translational machinery in each fraction are indicated: no ribosome components (−), ribosome subunits (Subunits), monosome (Mono.), low-molecular-weight polysome fractions (LMWF), and high-molecular-weight polysome fractions (HMWF).