Fig. 4. Cofilin controls the stiffness and activation of T cells.

(A) Naïve and effector T cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for the indicated times. The cells were lysed directly in SDS loading buffer and analyzed by Western blotting with antibodies against the indicated proteins. The amounts of phosphorylated cofilin were normalized to those of total cofilin. The bar graphs show densitometric analyses of relative band intensities. Western blots are representative of three independent experiments. (B) AFM stiffness mapping of naïve T cells after 1-hour incubation on coverslips coated with poly-D-lysine (Naïve) or poly-D-lysine and anti-CD3ε (Activated). Results are representative of two independent experiments. (C) Knocking down cofilin in effector T cells is hypothesized to increase cell stiffness and decrease both immune synapse size and the extent of activation. (D) Stiffness mapping of effector T cells expressing cofilin-specific shRNA or control shRNA, which is shown as a density histogram as described in Fig. 1E. Results are representative of two independent experiments. (E) Effector OT-II T cells expressing control shRNA or cofilin-specific shRNA were incubated for 5 min with antigen-pulsed APCs. The cells were then analyzed by Western blotting with an antibody against phosphotyrosine. Blots were probed with SLP-76 as a loading control. Western blots are representative of two independent experiments.