Fig. 5

Ser5 phosphorylation enhances L-plastin recruitment to invadopodia in MDA-MB-231 cells. a Expression pattern of the transduced MDA-MB-231 cells expressing the different GFP-fused L-plastin constructs. Cells were plated on gelatin-coated coverslips for 24 h and stained using anti-cortactin (blue) and Alexa Fluor 594-conjugated phalloidin (red) to visualize F-actin. GFP signal was amplified using the Alexa Fluor 488-conjugated GFP booster. Scale bar: 20 µm. Areas of actin, cortactin and L-plastin co-localization are seen in the overlay as white dot-like structures (right column). The insets show a higher magnification of the boxed areas. b Quantification of cortactin and F-actin-containing punctae per cell was performed using single confocal slices of the ventral surface of cells. Results are expressed as means ± SEM of three independent experiments in which 60–80 cells per conditions were assessed. One way ANOVA comparing all four groups showed no significance. c Percentage of GFP-positive invadopodia. Results are expressed as means ± SEM of three independent experiments. One way ANOVA followed by Tukey’s multiple comparison test (*p < 0.05). d Co-localization of Ser5 phosphorylated L-plastin with actin and cortactin in MDA-MB-231 cells. Cells were plated onto gelatin-coated coverslips for 24 h and stained using anti-cortactin (blue) and anti-Ser5-P-L-plastin (red) antibodies, followed by Alexa Fluor 633-conjugated phalloidin (magenta) to stain F-actin. Arrowheads point to areas of co-localization of proteins, which are seen in the overlay as white dot-like structures (right column). Scale bar: 10 µm