Silencing Proteases in Glioma Figure 8. RT PCR and Western blot analysis of the molecules associated with integrin-mediated migration. (A) RT-PCR of 4910 and 5310 glioma xenograft cells transfected with scrambled vector (SV-sh), MMP-9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh) plasmid shRNAs was performed as per standard protocol described in materials and methods. Several genes were downregulated with MU-sh and MC-sh treatments in both the xenograft cells. Results presented are the representative images of three independent experiments. (B) Western blot analysis showing the effect of bicistronic constructs (MU-sh and MC-sh) on the protein expressions of FAK, p-FAK (Tyr397), cSrc, p-Src (Tyr527), Rac1, Arp2, F-actin, talin1, paxillin, p-paxillin (Tyr118), a-actinin, cofilin and vinculin in 5310 human glioma xenograft cells. The expressions of majority of the proteins associated with integrin-mediated migration were reduced with MU-sh and MC-sh treatments. n = 3. doi:10.1371/journal.pone.0011583.g008 promoter was used to construct the shRNA-expressing vectors. A pCDNA3-scrambled vector with an imperfect sequence, which does not form a perfect hairpin structure, was used as a control (SV-sh). MMP-9 expressing (M-fl) plasmids in pDNR-CMV vector were designed and constructed in our laboratory whereas uPAR expressing (U-fl) and cathepsin B expressing (C-fl) plasmids were purchased from Origene (Rockville, MD). Antibodies Several antibodies were used in various techniques such as Western blot, immunocytochemistry, immunohistochemistry and fluorescent-activated cell sorting (FACS) analysis while determin- ing the changes in protein expression associated with various treatments. Antibodies targeting MMP-9, b3 integrin, a6 integrin, a9 integrin, a9b1 integrin, PECAM-1, FAK, Phospho FAK (Tyr 397), cSrc, talin1, vinculin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); Phospho Src (Tyr 527), Arp2, paxillin, phospho paxillin, cofilin were obtained from Cell Signaling (Boston, MA); b1 integrin, a6b1 integrin, aVb3 integrin were obtained from Millipore (Billerica, MA); uPAR was obtained from R&D Systems (Minneapolis, MN); a-actinin was obtained from Sigma (St. Louis, MO); cathepsin B was obtained from Athens Research and Technology (Athens, GA); aV integrin, F- actin were obtained from Abcam (Cambridge, MA); aVb3 PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11583