Supplementary Figure 7: Mtb PtpA binds TAB3 and blocks it from binding K63-linked ubiquitin chains.
From: Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system
(a) Mtb PtpA interacts with TAB3 in the yeast two-hybrid assay. Yeast strains were transformed with indicated plasmid combinations in which TAK1-TAB2 interaction serves as a positive control. Left, high-stringency. Right, low-stringency. (b) Mutation of Mtb PtpA at D126A does not interfere with the binding between PtpA and TAB3-NZF. GST-tagged proteins (10 μg) were used to precipitate 10 μg of purified PtpA (D126A) protein. Precipitation of the PtpA mutant by GST-TAB3-NZF was eventually analyzed by immunoblot analysis. (c) The NZF domain of TAB3, but not its D690T and S691A mutant form, could precipitate ubiquitin. On the contrary, the T671D and A672S mutant form of the NZF domain of TAB2, but not its wild-type form, could precipitate ubiquitin. GST-tagged proteins (10 μg) were used to precipitate 10 μg purified PtpA (D126A) protein. (d) Mtb PtpA precipitates free ubiquitin (His6-Ub), but not Lys 63-linked ubiquitin (K63-Ub) chains, as analyzed by in vitro precipitation assay. (e) Mtb PtpA blocks TAB3-NZF from binding K63-Ub chains as shown in the in vitro precipitation assay. GST-tagged proteins (10 μg) were used to precipitate 2 μg of K63-Ub chains. Data are representative of one experiment with at least three independent biological replicates.
