FIGURE 2.

A. Schematic diagram depicting treatments of specific populations of NIH/3T3 cells. HuR-FLAG: transfection (using Effectene transfection reagent, Qiagen) with a plasmid expressing FLAG-tagged HuR; -: transfection with pcDNA3 empty vector. Efficiency of transfection was ~30%–40%. Twenty-four hours after transfection all cells were serum-starved for 24 h in media containing 0.3% bovine serum. Where indicated by “serum stim”, cells were stimulated with 20% bovine serum for 45 min. B. Cells treated as described in A were scraped in PBS and the populations indicated in brackets in A were mixed just before lysis. Lysis, immunoprecipitation, and analysis of the samples were carried out as described in Figure 1 ▶, except that after immunoprecipitation, the same amount of in vitro transcribed β-globin RNA was added to each sample to control for differences in recovery of the RNA during the subsequent steps. Upper panel: RNase protection assay performed with a probe hybridizing to the last exon of the mouse c-fos RNA and a probe complementary to the exogenously added β-globin RNA. First two lanes: undigested probes (0.025% loaded) and digested probes (100% loaded) respectively. Lower panel: Western blot using the 3A2 monoclonal antibody against HuR. Note that under the conditions used, c-fos mRNA from non-stimulated cells is not detectable (data not shown).