Plasmids, small interference RNAs (siRNAs) and transient transfection. The plasmids pSG5-AR and mouse mammary tumor virus promoter-luciferase (MMTV-luc) were kindly provided by Dr. C. Chang (Cancer Center, University of Rochester, Rochester, NY) and the SV40-Renilla luciferase reporter plasmid (pRL) was bought from Promega Company. The plasmid for mammalian Angiotensin (1-7) and in vitro translation of CMTM3 was constructed using pcDNA3.1-myc-his b (?) (Invitrogen) expression vectors. CMTM3 was also subcloned into pEGFP-N1 plasmid (CLONTECH) and designated as pEGFP-CMTM3.
Three double-stranded siRNAs siCMTM3-1 (5′-AGCAGAGCCUUGUCUGUAU-3′), siCMTM3-2 (5′-CAAUGACCCUUCCAAGAAU-3′), and siCMTM3-3 (5′-GCCCUCAUCUACUUUGCUA-3′) targeting the nucleotides of the CMTM3, were synthesized by Genechem Corporation (Shanghai, China). A control double-stranded siRNA named as non-silencing (5′-UUCUCCGAACGUGUCACGU-3′), which had no sequence homology to any known human genes, was also generated.
View User's Journal
The role of processing in Rap downstream signaling
 |
