Calaluce et al. BMC Cancer 2010, 10:126 http://www.biomedcentral.com/1471-2407/10/126 Page 8 of 15 Figure 4 Validation of target CALM2 and CD9 mRNAs by quantitative RT-PCR. Quantitative RT-PCR using cell lysates, HuR antibody, and IgG1 from RIP-CHIP analysis confirmed results identifying CALM2 mRNA (A) and CD9 mRNA (B) as HuR targets. Change in gene expression is represented as fold increase in HuR immunoprecipitation as compared to IgG1. GAPDH mRNA was used as an endogenous control. Error bars represent SEM. p value is < 0.005. Experiments were done in triplicate (n = 3). further confirmed HuR binding to CD9 and CALM2 mRNAs by biotin pull-downs. The relevant portion of the mRNA was transcribed with biotin tags and incubated with lysates from the two cell lines to probe for interac- tions with protein. The mixture was then separated by pull-down using streptavidin-coated beads and HuR levels were analyzed by Western blot analysis. As seen in Figure 5, HuR specifically interacts with CD9 and CALM2 mRNAs in the 3’UTR regions, but not within the coding region (CR) or with a control biotinylated RNA corre- sponding to the 3’UTR of the housekeeping control GAPDH mRNA, which is not a target of HuR. HuR differentially regulates CD9 and CALM2 in MB-231 and MCF-7 cell lines To gain insight into the biological effects of these asso- ciations, we studied the consequences of stably increas- ing or decreasing HuR abundance. Individual MB-231 clones which over- and under-express HuR were estab- lished by limiting dilution (Figure 6A and 6B). MB-231 cells over-expressed HuR by about 140% (Figure 6A). HuR knock-down using lentiviral shRNA resulted in ~95% reduction in HuR expression (Figure 6B). Surpris- ingly, over-expression of HuR in MB-231 cells caused decreases in both CD9 protein and mRNA levels