miRNAs regulate the expression of PRKAR2B, KLHL5, and CLOCK by binding to their 3′-UTR. Cells expressing the protein of interest were transfected by the candidate miRNA and assessed for protein knockdown. The negative miRNA control has a scrambled sequence that does not repress any gene. Each panel shows a Western immunoblot (above) and the mean of at least 3 experiments below. Protein levels were normalized to levels of GAPDH. (A) Pre-miR-200b was transfected into Meg-01 cells (5 × 10⁶ cells), and lysates were harvested at 48 hours. The bar graph represents the mean 21% knockdown of 3 independent experiments. (B) Pre-miR-495 was transfected into Meg-01 cells (5 × 10⁶ cells), and lysates were harvested at 72 hours. The bar graph represents the mean 18% knockdown of 6 independent experiments. The miR-495 knockdown of KLHL5 showed a trend (P = .07). (C) Pre-miR-107 was transfected into HCT-DK cells (0.2 × 10⁶ cells), and lysates were harvested at 48 hours. The bar graph represents the mean 37% knockdown of 4 independent experiments. (D) The 50% decrease in GFP expression when the PRKAR2B 3′-UTR plasmid is cotransfected with pre-miR-200b compared with negative control in HCT-DK cells. Similarly, there was a 23% decrease in GFP when KLHL5 3′-UTR was cotransfected with pre-miR-495 (E) and 50% decrease in GFP with CLOCK 3′-UTR was cotransfected with pre-miR-107 (F). α-Tubulin was used as an internal control to normalize the GFP levels between the test and the control lanes.