Figure 4.

Effect of RAB27A (Asp7Tyr) mutation on NK cell degranulation (A) Co-immunoprecipitation of T7-MUNC13-4 and FLAG-RAB27A (WT or Asp7Tyr) in COS-7 cells, using anti-FLAG M2 magnetic beads. Co-immunoprecipitated T7-MUNC13-4 and immunoprecipitated FLAG-RAB27A were detected by immunoblotting with the antibodies indicated. (B) Ex vivo NK (CD3-CD56+) cells were either unstimulated in medium or stimulated with K562. Degranulation was measured by surface expression of CD107a. (C) Percent CD107a positive cells (normalized against medium only) relative to healthy controls (gray area). (D) NK cell degranulation of pre-activated NK cells (PHA (1μg/ml) and IL-2 (200U/ml) for 72 hrs), with either medium alone or K562 cells. (E) Percent CD107a positive pre-activated (as in D) NK cells (normalized against medium only) relative to healthy controls (gray area). (F) NK cell cytotoxicity measured by Cr⁵¹ release assay after 4hrs incubation. m, mutated; +, wildtype.