Journal: The Journal of Biological Chemistry

Article Title: Metalloprotease-Disintegrin ADAM12 Expression Is Regulated by Notch Signaling via MicroRNA-29 *

doi: 10.1074/jbc.M110.207951

Figure Lengend Snippet: caNotch1 increases Adam12 mRNA stability. A, diagram of gene reporter constructs. The indicated fragments of the promoter region of the mouse Adam12 gene were cloned into pGL4 luciferase reporter vector. The transcription start site is located at position +1, the translation initiation site is at position +220, the 5′-UTR region in the first exon is shown in black, and the genomic upstream sequence is shown in gray. B, NIH3T3 cells were co-transfected with the indicated luciferase reporters, pRL-TK, and either NICD1 or empty pcDNA3.1 vector. Firefly luciferase activity, normalized to Renilla luciferase, was measured 24 h after transfection, and -fold activation by NICD1 was calculated. The data represent the means ± S.E. from 2–4 independent experiments, *, p < 0.05. C, NIH3T3 cells stably transfected with pA12Luc.1 reporter were co-cultured with CHO-Dll1 or CHO-V cells. Firefly luciferase activity was measured 24 h later (relative luminescence units (RLU)). The data represent the means ± S.E. from 2 independent experiments. n.s., non-significant. D, NIH3T3 cells were infected with caNotch1 or control retroviruses. Actinomycin D was added 24 h after infection (time 0). The level of NICD1 at the indicated times after adding actinomycin D was evaluated by Western blotting using the Val1744 antibody. Adam12 and Gapdh mRNA levels were evaluated by semiquantitative RT-PCR. E, the band intensities in panel D were quantified by densitometry using the ImageJ software. The -fold change in Adam12 mRNA, normalized to Gapdh, is shown; the Adam12/Gapdh mRNA ratios at time 0 are set as 1. The data represent the means ± S.E. from 3 independent experiments.

Article Snippet: NIH3T3 cells grown in 6-well plates were co-transfected at 50% confluence with 0.5 μg of pA12.Luc reporters and NICD1 or empty pcDNA3.1 vector, 4×CSL reporters (Diane Hayward, Johns Hopkins University), and Igκ 2 -INF, a reporter containing two copies of the NF-κB binding site upstream of the interferon-β minimal promoter (Addgene plasmid 14886), together with 0.05 μg of Renilla luciferase vector (pRL-TK), using FuGENE 6 transfection reagent.

Techniques: Construct, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Activation Assay, Stable Transfection, Cell Culture, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Software