CGA-mediated inhibition affects firefly luciferase activity and protein levels, but does not result in reduced luciferase mRNA. Analysis of firefly luciferase protein, mRNA, and activity in X4F-reporter constructs containing RGS-(His)6-(Arg)8 insertions, from yeast strains bearing the indicated constructs (lanes 1–10). Firefly luciferase protein was detected with antibody to RGS-(His)6, with antibody to enolase serving as an internal control. In lanes 11–14, the top two panels contain dilutions of crude extracts expressing the X4F- RGS-(His)6 (AGA)8 reporter construct (10, 7.5, 5.0, 2.5 μg). Firefly luciferase mRNA was measured by RT-PCR analysis using primers homologous to bases 21–46 and 148–173, as indicated on the diagram below the panel; sequences are given in Supplemental Table S3; measurements of Actin mRNA served as an internal control. In the third panel, lanes 11-14 contain RT-PCR reactions in which dilutions of DNase-treated RNA from X4F-RGS-(His)6 (CGA)8-containing sample (0.6, 0.3, 0.15, 0.075 μg) were used in the RT reactions, with equal volumes of the RT reaction in each PCR reaction. Reverse transcriptase reactions in lanes 1–10 contain 0.3 μg total RNA input. Firefly luciferase mRNA amounts were first normalized to the levels of Actin mRNA and then quantified relative to the wild-type X4F-reporter construct with no insertion; the FLuc mRNA levels in the wild-type strain were arbitrarily set to 100. The presence of RGS-(His)6 is indicated by an asterisk (*). Firefly luciferase activity of X4F-reporter constructs containing RGS-(His)6-(Arg)8 insertions was normalized to the wild-type X4F-reporter construct with no insertion.
