Figure 3

LIMK1 regulates retinoid receptor function. A and B) Jurkat cells were transfected with 5.0 ug TATADR1-Luc in the presence of 2.5 ug and 5.0 ug of indicated plasmids. Cells were harvested after 36 h and luciferase activity measured as described in Experimental Procedures (A). Lysates were also subject o Western blotting using antibodies to anti-V5 tag (1:2,000) and beta-actin (1:10,000) (B). C) Jurkat cells were transfected with 5.0 ug of SP1-Luc plasmid in the presence of 5 ug of indicated plasmids. Cells were harvested after 36 h and luciferase activity measured as described in Experimental Procedures. SP1-Luc activity obtained in the presence of vector was normalized to 100%. D and E) Jurkat cells were transfected with LIMK1/WT, and CFL1/D460A plasmids and stained with anti-V5 Tag antibodies followed by treatment with F-actin stain. The cells were visualized using immunofluorescent microscopy (D) and the densitometric mean intensity of F-actin from V5-LIMK1 positive cells was quantified and compared to non-V5-LIMK1 expressing control cells (E) using AxioVision software from Zeiss Imager D1 fluorescent microscope. Bar, 2 um.