Fig. 3

α-Actinin-2 mediates the polymerization and selective aggregation of Actin at filopodia. A AGS cells were transfected with GFP or GFP-α-Actinin-2 for 24 h, then the cells were stained for GFP/GFP-α-Actinin-2 (green) and F-Actin (red). The fluorescence colocalization of α-Actinin-2 and F-Actin was performed using the ImageJ Plugins software. Manders′ Coefficient 1 (M1): the percentage of GFP/GFP-α-Actinin-2 colocalized with F-Actin, Manders′ Coefficient 2 (M2): the percentage of F-Actin colocalized with GFP/GFP-α-Actinin-2. B GFP-α-Actinin-2 or α-Actinin-2 was co-transfected with or without α-Actinin-2 siRNAs in AGS cells for 24 h, then the ratio of F-Actin/G-Actin was determined by G-Actin and F-Actin ratio assays (upper and lower panel), the input of total Actin, Tubulin, GFP-α-Actinin-2 and α-Actinin-2 were also showed in the middle panel. P: pellet; S: supernatant. C GFP-α-Actinin-2 or GFP was co-transfected with or without α-Actinin-2 siRNAs in AGS cells for 24 h, then cells were lysed for co-Immunoprecipitation (IP) assay by using α-Actinin-2 antibodies. D AGS cells were transfected with α-Actinin-2 for 24 h, then the membrane extract (PM), cytoplasmic extract (Cyto) and nuclear extract (Nu) of cells were separated by the Subcellular Protein Fractionation Kit. Immunoprecipitation (IP) assays were performed in PM, Cyto and Nu by using α-Actinin-2 antibodies. E GFP-α-Actinin-2 was co-transfected with or without α-Actinin-2 siRNAs in AGS cells for 24 h, then cells were treated as in (B) to separate the supernatant and pellet. The supernatant was used for co-Immunoprecipitation (IP) assay by using α-Actinin-2 antibodies. 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