Fig. 4

α-Actinin-2 enhances F-Actin binding ability by replacing α-Actinin-1 to form α-Actinin-2:α-Actinin-4 complexes. A Tissue and cell lysates were harvested and subjected to Western blotting using indicated antibodies. B AGS cells were transfected with GFP-α-Actinin-2 for 2 days following transfection with NC or α-Actinin-1/4 siRNAs for 24 h. The number of filopodia per cell and the average length of filopodia were quantified. The bars indicate the SD. The results are expressed as the mean ± SD of three independent experiments. *P < 0.05 using Ordinary one-way ANOVA. C AGS cells were transfected with GFP or GFP-α-Actinin-2 for 24 h, then the cells were stained for α-Actinin-1 (Cyan), GFP/GFP-α-Actinin-2 (green), α-Actinin-4 (magenta) and F-Actin (red), the fluorescence colocalization between α-Actinin-1/2/4 and F-Actin was performed using ImageJ Plugins software. Manders′ Coefficient 1 (M1): the percentage of P1 colocalized with P2, Manders′ Coefficient 2 (M2): the percentage of P2 colocalized with P1. D AGS cells were transfected with α-Actinin-2 for 24 h, then IP assays were performed using α-Actinin-1/2/4 antibodies. E and F In vitro G-Actin/F-Actin and α-Actinin-1:α-Actinin-4 complex or α-Actinin-2:α-Actinin-4 complex binding studies were performed and analyzed by Western blotting using the indicated antibodies