Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 Page 9 of 15 Figure 5 Biotin Pull-down of CD9 and CALM2. A. Scheme of Coding region (CR) and 3’UTR fragments for biotin pull-down assay. The sequences were obtained from Entrez data base. CR and 3’UTR fragments selected for amplification by PCR are as noted. B. 1% agarose gel electrophoresis showing PCR amplified products of the coding regions and 3’UTR’s for CD9 (442 bp and 432 bp, respectively) and CALM2 (443 bp and 610 bp, respectively). C. Biotin pull-down assay using lysates prepared from MB-231 cells. The binding of HuR to biotinylated 3’UTR transcripts from both CD9 and CALM2 mRNAs was specific. HuR did not bind a biotinylated control (GAPDH 3’UTR); and did not bind to biotinylated transcripts spanning the CR of CD9 or CALM2. Experiments were done in duplicate (n = 2). (Figures 6C and 6D). HuR knock-down, however, resulted in increases in both CD9 mRNA and protein levels (Figures 6C and 6E). This is the opposite of what we predicted, since HuR is generally regarded as a stabi- lizer of mRNA. In contrast, over-expression of HuR in MB-231 cells did not significantly alter the levels of CALM2 mRNA (Figure 6D). Figure 6F depicts a graphi- cal analysis which reveals that HuR over-expression decreases both CD9 mRNA and protein levels, as com- pared to controls (dashed line set at 100%). Whereas, HuR shRNA knock-down results in increases in both CD9 mRNA and protein levels above control levels. We performed similar analyses with MCF-7 cells, though the over-expression levels of HA HuR were only approximately 10%, since this was a pooled population (we have been unable to obtain MCF-7 clones which over-express HuR). In contrast, we generated MCF-7 clones with reduced HuR levels (93%) using lentiviral shRNA (Figure 7B). Western blot analysis of MCF-7 cells with over-expression of HuR reveals modest increases in CD9 protein levels (Figure 7C). There are also modest decreases in CD9 protein expression in MCF-7 with reduced HuR levels (Figure 7C). mRNA levels of CD9 and CALM2 are essentially unchanged in MCF-7 cells which over-express HuR (Figure 7D). As expected, HuR knock-down in MCF-7 cells using lenti- viral shRNA resulted in significant reductions in both CD9 and CALM2 mRNA levels (Figure 7E). The right panel in Figure 7E indicates efficiency of HuR mRNA knock-down which is consistent with the protein data (Figure 7B). These results are summarized in Figure 7F. There are no significant changes seen in CD9 mRNA