FIGURE 8.

Full-length FLNa chimera containing CH1 of α-actinin1 is resistant to ASB2α-mediated degradation and rescues spreading in filamin-deficient cells. A, CHO cells transfected with full-length FLNa GFP and CH1α-FLNa GFP were fixed and stained for phalloidin (Phall). Scale bar, 10 μm. B, cells from A were lysed and blotted using anti-GFP antibody. C, CHO cells transiently expressing full-length FLNa GFP, CH1f-CH2α GFP, CH1α-CH2f, and CH1α-FLNa GFP were transfected with either dsRed-ASB2α or dsRed-ASB2αΔS, and degradation of the GFP protein in ASB2α-expressing cells was assessed 48 h later as in Fig. 1 (see “Experimental Procedures” for details). Data are from at least four independent experiments. D, HT1080 WT, FLNabKD, or FLNabKD cells transiently transfected with CH1α-FLNa GFP or FLNaΔABD 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. Areas were normalized to the mean WT area and presented as mean.± S.E. from three independent experiments (>16 cells per condition). p values were calculated using the t test. E, HeLa cells were co-transfected with dsRed-ASB2α and GFP (dsRed-ASB2α + GFP), dsRed-ASB2α and CH1α-FLNa GFP (dsRed-ASB2α + CH1α-FLNa GFP), or dsRed and CH1α-FLNa GFP (dsRed + CH1α-FLNa GFP). 48 h after transfection, cells were detached and re-plated on fibronectin-coated coverslips. 3 h after plating, cells were fixed, and the areas dsRed and GFP double-positive cells were measured and compared with the nontransfected cells. Pixel = 0.416 μm². Dot plot shows the overall population distribution from four independent experiments; dotted line shows the mean area, and the box and whiskers plots show quartiles. p values were calculated using the t test.