Fig. 2. L-plastin phosphorylated on Ser5 localises to the actin cytoskeleton in Vero cells. Transfected Vero cells expressing wild-type L-plastin or S/A variant were processed for immunofluorescence staining. (A) Intracellular distribution of L-plastin phosphorylated on Ser5. (Upper row) Cells expressing wild-type L-plastin (WT) were stained with Rhodamine-phalloidin (left panel) and anti-Ser5-P antibody (middle panel). Alexa-Fluor-488-coupled anti-rabbit IgG antibody served as secondary antibody. Merged image of enlarged regions (boxes) of left and middle images is shown on the right. Red, F-actin; green, anti-Ser5-P antibody. (Lower row) Cells expressing wild-type L-plastin were double-stained with anti-sv-tag antibody (left panel) and anti-Ser5-P antibody (middle panel) with Alexa-Fluor-594-coupled anti-mouse and Alexa-Fluor-488-coupled anti-rabbit IgG antibody as secondary antibodies, respectively. Merged image of enlarged regions (boxes) of left and middle images is shown on the right. Red, total WT L-plastin; green, phosphorylated WT L-plastin. Bars, 15 µm. (B) The anti-Ser5-P antibody does not react with Ser5Ala L-plastin. Cells expressing the non-phosphorylatable Ser5Ala variant (S/A) were double-stained with anti-sv-tag antibody (left) and anti-Ser5-P antibody (right) as described above. Bar, 15 µm.