C4 domain in TRBP is required for colocalization with endogenous Dicer. HeLa cells were transfected with 0.5 μg of pCMV-Myc-TRBP1 or pCMV-Myc-TRBP2 (A), pCMV-Myc-TRBP1ΔC4 or pCMV-Myc-TRBP2ΔC4 (B), as indicated on the left. Cells were fixed and labeled with mouse anti-Myc and rabbit anti-Dicer (pAb349) followed by anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 546. The cells were mounted and assayed for fluorescence by laser scanning confocal microscopy on a Zeiss LSM Pascal 5 microscope. Alexa Fluor 488 and Alexa Fluor 546 fluorescences are represented as green and red, respectively, while merged colors appear as yellow. Images showing the colocalization of the two proteins detected by the yellow dots or absence of colocalization are amplified on the right. C) TRBP2 and TRBP2ΔC4 do not colocalize. HeLa cells were transfected with 0.5 μg pEGFP-C1-TRBP2 and either pCMV-Myc-TRBP2 or pCMV-TRBP2ΔC4. Cells were fixed and labeled with anti-Myc followed by anti-mouse Alexa Fluor 546. The cells were mounted and assayed as previously. EGFP signal is shown in green and Alexa Fluor 546 fluorescence is represented in red. Images showing colocalization of the two proteins detected by yellow dots, or absence of colocalization, are amplified of the right.