Expression of functionally overexpressed or endogenous gelsolin and its effects on cortical F-actin. (A) Left, Western blot of gelsolin-EGFP expression in CEM.NKR-CCR5 cells. Gelsolin-EGFP fusion protein was stable and did not undergo any processing of its C-terminal EGFP tag. α-tubulin and pEGFP-N1 are controls for total protein and free EGFP, respectively. Right, Quantification of gelsolin-EGFP (45.2%) and free EGFP (56.9%) expressing cells by flow cytometry. One representative experiment of three is shown. (B) Left, a series of confocal images, xy midsections, showing the distribution of overexpressed gelsolin-EGFP. F-actin, free EGFP and merged images for F-actin/gelsolin-EGFP co-localization at cell-surface are shown. One representative experiment of three different experiments is shown. Right, flow cytometry analysis of F-actin in EGFP- (control, taken as 100% F-actin expression) or gelsolin-EGFP- expressing cells. Data are mean ± s.e.m of three independent experiments: *p < 0.05, Student’s t-test. (C) Left, line scans show the fluorescence intensity profiles of cortical F-actin and the overexpressed free EGFP or gelsolin-EGFP proteins. Images correspond to the results presented in the merged images in panel B, analysed along the horizontal line drawn in panel C. Right, quantification of the fluorescence intensity profiles of cortical F-actin in a series of cells (12 cells per series) analysed by line scanning confocal microscopy (8 cortical points analyzed per cell). Data are mean ± s.e.m. of three independent experiments: **p < 0.01, Student’s t-test. (D) Left, a series of confocal images showing the distribution of endogenous gelsolin. F-actin and merged images for F-actin/gelsolin co-localization at cell-surface are shown. One representative experiment of three is shown. Right, Western blot analysis of endogenous gelsolin and F-actin expression. α-tubulin is a control of total protein expression. One representative experiment of three is shown. In B, C and D, scale bar = 5 μm.