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. 2019 Jun 10;129(7):2717–2729. doi: 10.1172/JCI126341

Figure 5. Dok3 mediates Card9 dephosphorylation through PP1 recruitment.

Figure 5

(A) Purified Dok3+/+ and Dok3–/– neutrophils were treated with zymosan (10 μg/ml) for 30 minutes. Whole cell lysates (WCL) were IP with a Card9-specific antibody, and precipitates were probed for p-Thr and Card9. Whole cell lysates were also probed for Card9 and β-actin. (B) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for 30 minutes, and IP with a Dok3-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Card9, PP1, PP2A, and Dok3. (C) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated for 30 minutes with zymosan (10 μg/ml), and IP with a Card9-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Dok3, PP1, PP2A, and Card9. (D) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for 30 minutes, and IP with a PP1-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Card9, Dok3, and PP1. (E) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for indicated periods of time and IP with a Dok3-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for Card9, PP1, and Dok3. (F) Co-IP analysis of cell lysates from purified Dok3+/+ and Dok3–/– neutrophils treated with zymosan (10 μg/ml) for 30 minutes, and IP with a Card9-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for PP1, Dok3, and Card9. (G) Co-IP analysis of cell lysates from purified Dok3+/+ neutrophils treated with zymosan (10 μg/ml) for 30 minutes and IP with a Card9-specific antibody or an IgG control. Precipitates and whole cell lysates were probed for PP1α, PP1β, PP1γ, Dok3, and Card9. All images are representative of 3 independent experiments.