Figure 3

Coro1A modulates PKCθ- mediated subcellular location in activated T cells. Jurkat cells were transfected with GFP inert protein control, PKCθ or Coro1A wild-type cDNA expression plasmids, respectively. (A) Co-localization of transfected PKCθ and Coro1A occurred at a rate of approximately 76% in intact cells as measured by immunofluorescence. Jurkat T cells were stimulated for 20 min with anti-CD3/anti-CD28 antibodies and analysed by subsequent staining with protein-specific antibodies. A representative image is shown. (B) Translocation of PKCθ to the plasma membrane is inhibited by overexpression of Coro1A. Jurkat cells were transfected with Coro1A or GFP, respectively. After 21 hrs cells were stimulated with anti-CD3/anti-CD28 antibodies for 20 min or left unstimulated, as indicated and subcellular distribution of endogenous PKCθ was determined by immunoblotting. The cell fractions are defined as the soluble (s) fraction, the particulate (pt) fraction and the Triton-X100 non-soluble (ns) fraction, which were prepared as described in the Additional file 1 (Supplementary Methods). The p59 fyn protein was detected to control for cell fractionation. (C) Lipid rafts were prepared by fractionation of sucrose gradients and immunostained for PKCθ and Coro1A. As a marker for the raft fraction, the marker ganglioside M1 of each fraction was quantified in a dot blot employing HRP-Choleratoxin B (not shown). A representative experiment of 3 independent experiments is shown.