Prl sv40 renilla control vector

Luciferase reporter vectors (see above) and 10 ng of the pRL-SV40 (Renilla) control vector (Promega), and 100 nM miR-302b or scrambled sequence miRNA control (Ambion Inc, Austin, TX) were co-transfected into MDA-MB-231 cells in 12-well plates. Firefly luciferase activity was measured with the Dual Luciferase Assay Kit (Promega) 24h after transfection and normalized for the Renilla luciferase reference plasmid. Reporter assays were carried out in at least in quadruplicate and the mean (representative of at least two independent experiments) ± S.D. was reported. Statistical significance was analyzed by the unpaired Student t-test.

Luciferase gene reporter assays were carried out in 24-well plates. In brief, subconfluent mIMCD-3 cells were co-transfected with pGL3 control vector or pGL3–24p3/Lcn2-Luc containing murine Lcn2 gene promoter (Addgene Inc., ID 25463) in combination with plasmids expressing protein of interest. pRL-SV40 Renilla control vector (Promega) was co-transfected in each condition. The luciferase activity was measured 36 h after transfection using a dual-luciferase reporter assay system (Promega) as recommended. Luciferase activity in each cellular lysates was normalized to Renilla luciferase activity. Results are presented as changes of transactivation of the murine LCN2 promoter constructs relative to the original activity of this promoter constructs in cells transfected with empty vector control plasmid.