Expression and targeting of IL-2 fused to different membrane anchors of CD16b.

<p>(<b>A</b>) Surface expression levels of IL-2 fused to different membrane anchors on HEK-293 cells. HEK-293 producer cells were transfected with indicated plasmids and stained with IL-2-specific (black line) or non-binding control mAb (grey histograms), followed by flow cytometric analysis. Numbers indicate percent positive cells. (<b>B</b>) Percentage of IL-2 positive HEK-293 cells upon transfection with the indicated constructs. (<b>C</b> and <b>D</b>) Expression of IL-2v in HEK-293 producer cells and corresponding VNP preparations as detected with IL-2-specific antiserum. Anti-Gag was used as loading control. Bottom panels show quantification of band intensities (Image J 1.48 software) normalized to values obtained from whole cell lysates of IL-2::GPI transfectants and corresponding VNP. (<b>E</b>) Relative amounts of IL-2 targeted to VNP compared to IL-2::GPI VNP. (<b>F</b>) GPI-anchor attachment targets IL-2 into lipid rafts of HEK-293 cells. Triton X-100 lysates of HEK-293 cells were resolved by 5 to 40% sucrose gradients into nine fractions (top to bottom), subjected to SDS-PAGE, blotted and probed (IB) with IL-2-specific antiserum, CD59 (lipid raft targeting control) and CD147 (non-lipid raft targeting control) mAb. (<b>G</b>) Dot-blot analyses of relative amounts of particles secreted by HEK-293 producer cells transfected with indicated constructs using p30Gag immunoblotting (IB). Data are representative (A, C, D, F) or show the summary (B, E, G) of three (except six for IL-2::GPI and IL-2::1IgGPI) (A to D), four (E, G) and two (F) independent experiments. * p < 0.05, ***, p < 0.001; Kruskal-Wallis test and Mann-Whitney-U-test followed by post hoc Bonferroni correction (B), ANOVA and Tukey’s multiple comparison test (G).