TCTTAAACATTGTTTTGTAGTGTATATTACTTGTCCATTCCTTTAAGGGGAGCTTTAAA CACTCTTTTGTAGATTACTTTTGGGGGATATATTTTGAGAATGATGAAACGGAATAAAATTGTAAAAAATTAATTGTAGTTTTAA.
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.
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.