DGCR8 expression is regulated by the Microprocessor. Hela cells were transfected with either scrambled (Sc), DGCR8, or Drosha siRNA as indicated. Cells were collected for analysis 60 h post-transfection. (A) Whole cell extracts were analyzed by Western blot using anti-Drosha, DGCR8, and Tubulin antibodies. (B) Total RNA was analyzed by quantitative RT-PCR using primers specific for Drosha, DGCR8, and Actin mRNA. Error bars represent SEM with N = 3. (C) Schematic representation of the firefly luciferase reporter containing DGCR8 5′UTR (pGL3c DGCR8 5′UTR). (D) Hela cells were co-transfected with either pGL3c or pGL3c DGCR8 5′UTR plasmid together with a TK-Renilla luciferase plasmid. Luciferase activity was measured 48 h post-transfection. Firefly luciferase activity was normalized relative to that of Renilla luciferase. Error bars represent SEM with N = 3. (E) Hela cells were transfected with pGL3c DGCR8 5′UTR together with TK-Renilla luciferase vector, and either scrambled, DGCR8, or Drosha siRNA as indicated. Luciferase activity was measured 60 h post-transfection. Renilla luciferase activity was used for normalization. Error bars represent SEM with N = 5.
