Figure 7

TRBP1 and TRBP2, but not TRBPsΔC4 rescue the RNAi pathway in U251MG cells. A) TRBP1 and TRBP2 rescue the RNAi pathway in U251MG cells. Human U251MG cells were transfected with none or 1 μg of EGFP and 4 μg of either non-silencing (ns) or anti-GFP (gfp) sh- or miRNA, as indicated, and 1 μg of pcDNA3 (left panels), pcDNA3-TRBP1 (middle panels) or pcDNA3-TRBP2 (right panels). (Top panels) EGFP knock-down was assessed by fluorescence, and average percentages of EGFP knockdown were calculated from luminosity intensity values of four fields per condition. Representative photos are shown. (Bottom panels) Cell extracts were analyzed by immunoblotting against EGFP and actin. Percent decrease of EGFP signal was calculated by densitometry analysis. B) Myc-TRBP1 and TRBP1ΔC4 are equally expressed in U251MG cells. U251MG cells were transfected with 1 μg of either pCMV, pCMV-Myc-TRBP1 or pCMV-Myc-TRBP1ΔC4 as indicated. Cell extracts were analyzed by immunoblotting against Myc and actin. C) TRBP1ΔC4 and TRBP2ΔC4 do not rescue the RNAi pathway in U251MG cells. U251MG cells were transfected with 1 μg of EGFP and 4 μg of non-silencing (ns) or anti-EGFP (gfp) shRNA as indicated, and 1 μg of either pCMV, pCMV-Myc-TRBP1, pCMV-Myc-TRBP1ΔC4, pCMV-Myc-TRBP2 or pCMV-Myc-TRBP2ΔC4 as indicated. (Top panels) EGFP knock-down was assessed by fluorescence, and average percentages of EGFP knockdown were calculated from luminosity intensity values of four fields per condition. Representative photos are shown. (Bottom panels) Cell extracts were analyzed by immunoblotting against EGFP and actin. Percent decrease of EGFP signal was calculated by densitometry analysis.