Munc13-4 is transiently recruited in membrane rafts after NK-cell activation. Primary NK cells were fractionated by sucrose gradient centrifugation. An equal amount of proteins recovered from the raft and detergent-soluble fractions were analyzed by Western blot. Unstimulated samples (−) were treated as described in “Methods.” (A) The purity control of the raft and soluble fractions was performed by immunoblotting as indicated. One representative experiment is shown. (B) Cells were stimulated with anti-CD16 mAb or treated with PMA/ionomycin for the indicated times. Immunoblot analysis of raft and soluble fractions was performed. The percentage of raft-associated Munc13-4 was obtained by densitometric analysis evaluating the ratio of raft-associated Munc13-4 with respect to the total amount of Munc13-4 extrapolated by the sum of Munc13-4 levels in the single fractions normalized for the loaded volume (means ± SD, n = 3). (C) Cells were stimulated for 10 minutes with the indicated mAbs. The percentage of raft-associated Munc13-4 was evaluated as in panel B (means ± SD, n = 5). Differences between unstimulated and CD16-, DNAM-1-, 2B4/DNAM-1-, and 2B4/NKG2D-stimulated cells were significant (*P < .05; **P < .01). (D) In the same experiment described in panel B, cells were subjected to hypotonic shock for K⁺ depletion or treated with Dynasore and CD16 stimulation was performed as above. One representative experiment of 3 performed is shown. The percentage of raft-associated Munc13-4 was evaluated as above (means ± SD, n = 3).