Figure 4

Effects of exo-mRNA fraction on reverse transcription (RT)-PCR for kallikrein 1b26 (klk1b26) mRNA and inhibition of klk1b26 translation by miRNA preparation from female submandibular glands (SMGs). (A) Effect of exo-mRNA fraction on RT-PCR with a primer pair targeting the 5'-terminal region of klk1b26 mRNA. RT-PCR was carried out with the primer pair F21 forward and R552 reverse in the presence or absence of the exo-mRNA fraction (100 ng/μl) from male or female SMGs. (B) Quantitative determination of density of klk1b26 PCR products in Figure 4A was measured by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/). Values are averages of the duplicate assay. (C, D) Inhibition of klk1b26 translation in vitro by miRNA preparation from female mouse SMGs. After the preincubation of mRNAs purified from male SMGs with miRNA preparation from male or female SMGs, the in vitro translation was performed and klk1b26 protein synthesized was analyzed as described in Methods. Representative results are shown. Similar results were obtained from three independent experiments (C). Quantitative determination of density of the [³⁵S]klk1b26 protein band. Values represent the mean ± SD (n = 3) of the relative density (D). (E) Representative autoradiograms of [³⁵S]methionine-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein on the SDS-PAGE gels. In vitro translation was performed as described in Methods. GAPDH protein was immunoprecipitated with anti-GAPDH antibody. (F) Quantitative determination of density of the [³⁵S]klk1b26 protein band. Values are the mean ± SD (n = 3) of the relative density.