Mir 484 mimic

TCTTAAACATTGTTTTGTAGTGTATATTACTTGTCCATTCCTTTAAGGGGAGCTTTAAACACTCTTTTGTAGATTACTTTTGGGGGATATATTTTGAGAATGATGAAACGGAATAAAATTGTAAAAAATTAATTGTAGTTTTAA.
Seventy-thousand cells were seeded in 24-well plates and co-transfected with 100 nM of mirVana miRNA mimic, negative control #1 (Cat# 4464058), or miR-484 mimic (Cat# 4464066, Assay ID MC10379) from Thermo, 100 ng of luciferase reporter pLenti-UTR-Luc PSMG1 WT or pLenti-UTR-Luc PSMG1 MUT, and 4 ng of pRL-CMV Renilla luciferase reporter (Cat# E2261) from Promega (Madison, WI), using TransIT-X2 (Cat# 6004) from Mirus (Madison, WI). Cells were cultured for another 48 h and washed once in PBS. Using a Luc-Pair Duo-Luciferase HS Assay Kit (Cat# LF004) from GeneCopoeia (Rockville, MD), cells were then lysed, loaded onto white 96-wells in quadruplicates, and their luciferase/Renilla ratios were measured using a FLUOstar Omega microplate reader from BMG Labtech. The experiment was repeated for n = 4.

DU145 cells were transfected with 30 nM of mirVana miRNA mimic, negative control #1 (Cat# 4464058), or miR-484 mimic (Cat# 4464066, Assay ID MC10379) from Thermo (Waltham, MA). Forty-eight hours after transfection, total protein was extracted from cells using RIPA buffer (Cat# 89901) with Halt Protease Inhibitor Cocktail (Cat# 87786), and 50 μg of protein was loaded on a 16% Tris-Glycine gel (XP00165BOX) from Thermo followed by immunoblotting. Primary antibodies used were PSMG1 (1:1000 of Cat# 13378) from Cell Signaling (Danvers, MA) and β-Actin (1:5000 of sc-47778) from Santa Cruz (Dallas, TX). The secondary antibody for the PSMG1 Western was an ECL anti-Rabbit IgG, HRP-linked F(ab')₂, fragment (from donkey) (#NA9340V) from GE Healthcare (Marlborough, MA), used at a 1:5000 dilution. Visualization of β-Actin using the labeled primary antibody from Santa Cruz does not require a secondary antibody. Chemiluminescent signal was captured on the Bio-Rad ChemiDoc Imaging System. The experiment was repeated for n = 3.