Figure 4

RNAi -mediated disruption of insulator function in the S2 assay. A. Diagram of 2xR transgenes used in RNAi knockdown tests. B. RT-PCR assessment of SuHw, GAF and dCTCF transcript level in S2 cells containing 2xR-insulator transgenes. Transgenes used in transfection are indicated on top of the panel. Double-strand RNA (dsRNA) used in knockdown treatment and mRNA-specific primers used in RT-PCR reactions are indicated on the left. S2 cells not treated (-, left lanes) or treated (+, right lanes) with dsRNA were used in RT-PCR using gene-specific primers and Actin 88f primers. The asterisk indicates the expected actin product at 370 bp. C. Western blot analysis of SuHw protein level in S2 cells. Left lane, molecular weight standard in kilodalton (KD); middle lane, untreated cells; and right, dsRNA-SuHw-treated cells. Arrowhead points to the position of SuHw at ~145 KD. Asterisk indicates a non-specific band reactive to the antibody. D. Summary of mRNA and protein reduction in the RNAi-mediated knockdown. N indicates the number of replicate of RT-PCR used in the assessment. E. Changes in GFP/RFP ratio as a result of knockdown (untreated cell = 100%). The dsRNA used in the knockdown is indicated below the bar graph. Number of replicates is indicated in parentheses.