Phrl null vector

The procedure for the luciferase reporter assay has been previously reported [18 (link),27 (link)]. RAW264.7 cells were plated in 96-well plates at 5 × 10³ cells per well. Cells were co-transfected using Lipofectamine 2000 with either WT or mutant pGL3-RANK constructs (200 ng) and miR-503 or miR-NC for 48 h. The Dual-Luciferase Reporter Assay System (Promega) was used to quantify luminescent signal by using a luminometer (Glomax, Promega). For each sample, the firefly luciferase activity value was normalized to the Renilla luciferase activity value from the co-transfected phRL-null vector (Promega).

For functional analysis of miR-497∼195, the segments of the mouse Fbxw7 and P4HTM 3′-UTR, including the predicted miR-497∼195–binding site, were PCR-amplified. The PCR products were purified and inserted into the XbaI-FseI site immediately downstream of the stop codon in the pGL3 control luciferase reporter vector (Promega Corp.), resulting in mouse WT-pGL3-Fbxw7 or WT-pGL3-P4HTM vectors. The Fbxw7 and P4HTM mutants for the miR-497∼195 seed regions were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) to get mouse MUT-pGL3-Fbxw7 or MUT-pGL3-P4HTM. Mouse BEPs were transfected with either WT or mutant pGL3 construct, the pRL-TK renilla luciferase plasmid (Promega Corp.), and agomiR-497∼195 or agomiR-NC for 48 h using Lipofectamine 2000 (Invitrogen). The dual luciferase reporter assay system (Promega Corp.) was used to quantify luminescent signal using a luminometer (Glomax; Promega Corp.). Each value from the firefly luciferase assay was normalized to the renilla luciferase value from the cotransfected phRL-null vector (Promega Corp.).

NIH3T3 cells were transfected for 24 h with either WT or mutant pMIR REPORT-Tnfrsf11a-3’-UTR constructs (200 ng) along with miR-218–2-3p using Lipofectamine 2000. The Dual-luciferase reporter assay system (Promega, Madison, WI) was used to quantify the luminescent signal using a luminometer (Glomax; Promega). Each value from the firefly luciferase assay was normalized to the Renilla luciferase assay value from the co-transfected phRL-null vector (Promega).