Supplementary Figure 5: TRAF6 activity is essential for the K63-linked ubiquitylation of Rac1. | Nature Immunology

Supplementary Figure 5: TRAF6 activity is essential for the K63-linked ubiquitylation of Rac1.

From: Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity

Supplementary Figure 5

(a) The distribution of Rac1 (red) in Mst1fl/flMst2fl/fl (WT) BMDMs, Mst1fl/flMst2fl/flLyz2-Cre (cDKO) BMDMs and WT BMDMs treated with NSC23766 after phagocytosis with GFP-E. coli. Images shown are representative of approximately 100 cells. Scale bar, 20 μm.

(b) Pull-down experiments were performed using purified Flag-Rac1WT, Flag-Rac1G12V or Flag-RacT17N incubated with lysates from LPS-treated BMDMs. Immunoblots show the association of TRAF6 with Flag-RacT17N.

(c) Ubiquitination and immunoprecipitation assay of 293T cells transfected with plasmids expressing Flag-Rac1, Myc-TRAF6 and HA-ubiquitin (Ub), HA-ubiquitin K48 (Ub 48) or HA-ubiquitin K63 (Ub 63). Cell lysates were immunoprecipitated with anti-Flag antibody. The cell lysates and immunoprecipitates were analysed by immunoblotting with antibodies as indicated. Anti-HA immunoblotting shows the level of Rac ubiquitylation.

(d) Knock-down of TRAF6 expression levels in BMDMs or RAW264.7 cells with different TRAF6 shRNAs.

(e) The ubiquitination levels of WT or mutated Rac1 with the indicated substitutions of lysine (K) with arginine (R) in 293T cells.

Data are representative of three independent experiments.

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