Figure 3. SETD6 is a transcriptional regulator. (A) HEK293 cells were seeded at a density of 100,000 cells per well of a 12-well plate and transfected the following day with 0.75μg of either pGL3 SV40luc (GAL4-unresponsive, i.e., G0) or pGL3 G5SV40luc (GAL4-responsive, i.e., G5) and 0.25μg GAL4DBD, GAL4-SETD6SET, or GAL4-SETD6WT. Values are expressed as relative luciferase units (R.L.U.) as measured 30 h post-transfection and error bars originate from the standard error of the mean from a representative experiment with each sample in quadruplets. *P values < 0.04. (B) As in panel A, but increasing amount (0.05 μg, 0.1 μg, 0.25 μg, and 0.5 μg) of GAL4-SETD6SET or GAL4-SETD6WT were transfected along with 0.5 μg reporter. *P values < 0.03 between the 0.0 5μg and 0.5 μg samples. (C) The indicated GAL4-tagged KMTs (0.25 μg) were transfected along with the G5SV40luc reporter (0.75 μg). *P values < 0.001. The expression level of each GAL4/FLAG-tagged constructs was assessed by denaturing PAGE of protein extracts followed by immunoblotting using the HRP-conjugated α-FLAG M2 or α-tubulin antibodies.