Impact of transfection of T-lymphoblasts with flotillin-2-G2A on cell polarization and capping of uropod components. (A,B) T-lymphoblasts were incubated for 22 hours in the absence of IL-2, followed by transfection with 2 μg of constructs encoding for HA-tagged wild-type flotillin-2 (flo2) or flotillin-2-G2A (flo2-G2A). Five hours after transfection, IL-2 was added followed by a further 17 hour incubation, stimulation with SDF-1 (15 minutes, 40 ng/ml), TCA-fixation and double-labeling for HA-tagged wild-type or mutated flotillin-2 (flo2-HA; flo2-G2A-HA) and endogenous flotillin-1 (flo1) (A) or HA-tagged wild-type or mutated flotillin-2 and endogenous PSGL-1 (B), using a polyclonal rabbit anti-HA antibody and monoclonal murine antibodies directed against flotillin-1 and PSGL-1. Bar, 10 μm. (C,D) T-lymphoblasts were treated as described for (A,B), and the amount of polarized transfected cells (n = 3) (C) or the amount of transfected cells with capped endogenous flotillin-1 (n = 4) or PSGL-1 (n = 3) (D) were evaluated (mean ± s.e.m.). 30-50 transfected cells were analyzed per sample and experiment. *P < 0.025, **P < 0.0125 for differences between data obtained for cells transfected with HA-tagged wild type or mutated flotillin-2.
Affentranger et al. BMC Cell Biology 2011 12:28 doi:10.1186/1471-2121-12-28