miR-34a directly represses DGKζ expression through targeting both DGKζ 3' UTR and CR.

<p>(<b>A</b>) Schematic reporter representation of construct to test the function of DGKζ 3' UTR. SV40, SV40 promoter. (<b>B</b>) Fluc reporter bearing DGKζ 3' UTR was cotransfected into 293T cells with Rluc reporter control and either miR-34a or empty plasmid (mock). Twenty-four hours later, luciferase activities were measured by a luminometer, and Fluc results were normalized by Rluc activities. Bar graphs represent mean ± SEM from three independent experiments (a.u., arbitrary unit; ***<i>P</i> < 0.001). (<b>C</b>) 293T cells were stably transfected with plasmid expressing human (hsa)- or mouse (mmu)-originated miR-34a, and a western blot was carried out for endogenous DGKζ protein. β-actin was used as a loading control. (<b>D</b>) 293T cells were transiently transfected with flag-tagged wild-type DGKζ (flag-DGKζ-WT), flag-DGKζ-MM bearing mutated seed match (CAC<u>T</u>GC<u>C</u> to CAC<u>C</u>GC<u>A</u>), a cotransfected flag-SLP76 as an internal control, and miR-34a plasmids as indicated combinations. Twenty-four hours after transfection, the cells were harvested, and a western blot was conducted using an anti-flag antibody. p38 was used as a loading control for total lysates.