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).