FIGURE 6.

Full-length FLNa K42R/K43R/K135R is resistant to ASB2α-mediated degradation and impaired in targeting to stress fibers. A, CHO cells transfected with full-length FLNa GFP and FLNa K42R/K43R/K135R GFP were fixed and stained for phalloidin (Phall). Scale bar, 10 μm. B, CHO cells transfected with full-length FLNa GFP and FLNa K42R/K43R/K135R GFP (K42/43/135R GFP) were lysed and immunoblotted using anti-GFP antibody. C, CHO cells transiently expressing full-length FLNa GFP, FLNaABD GFP, full-length FLNa K42R/K43R/K135R GFP, FLNaABD K42R/K43R/K135R GFP, and FLNaCH2 GFP were transfected with either dsRed-ASB2α or dsRed-ASB2αΔS, and FLNa degradation was assessed as described in Fig. 1 (see “Experimental Procedures” for details). Bar chart depicts mean percentage of GFP-tagged protein remaining ± S.E. Data are from at least six independent experiments. D, HT1080 wild-type (WT) and FLNa and FLNb double knockdown cells (FLNabKD) or FLNabKD cells transiently expressing either wild-type FLNa GFP or FLNa K42R/K43R/K135R GFP were plated on fibronectin-coated coverslips. 3 h after plating, cells were fixed, and areas were measured by manually rendering the cell contour in phase contrast and normalized to the size of untransfected WT cells. Bar chart depicts mean relative cell area ± S.E. for each condition from three independent experiments (>20 cells per condition). p values were calculated using the t test.