Mutation of S2152A within FLNa abolishes TCR-mediated T-cell adhesion, interaction with APCs and LFA-1 activation. (A) Jurkat T cells were transiently transfected with dsRed C1 vector (dsRed) or plasmids encoding wild type (WT) dsRed-tagged FLNa (FLNa WT) or dsRed-tagged S2152A or dsRed-tagged S2152E FLNa mutants (FLNa S215A and FLNa S2152E). After 24 h the expression of WT and its mutants were analyzed by anti-dsRed and FLNa immunoblotting. Detection of β-actin served as loading control. (B) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated for 30 min with CD3 antibodies. Cells were analyzed for adhesion to ICAM-1-coated 96 well plates. Bound cells were counted and calculated as percentage of input ( n = 4) (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001). (C) Cells were transfected as described in (A) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min at 37°C. The percentage of conjugates was defined as the number of double-positive events in the upper right quadrant ( n = 4) (mean ± SEM; *** p ≤ 0.001). (D) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated with anti-CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 to detect the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry and data are normalized against LFA-1 expression detected by MEM48 ( n = 4) (mean ± SEM; *** p ≤ 0.001). (E) HEK 293T cells were transfected with either dsRed, dsRed-tagged FLNa wild type (FLAa WT) or its mutants (FLNa S2152A and FLNa S2152E). 24 h after transfection, whole cell extracts were prepared and analyzed for the expression of dsRed and dsRed-tagged FLNa forms by Western blotting using the indicated antibodies (left panel). Lysates were incubated with GST-fusion proteins bound to glutathione-sepharose beads. Precipitates were analyzed by Western blotting using the indicated antibodies (right panel). One representative experiment of 3 is shown. (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001).