Fig. 1

Mtb Rv1468c directly binds to poly-Ub chains in a UBA-dependent manner. a, b Immunoblot analysis of whole bacterial lysate of Mtb cells pretreated with (+) or without (–) proteinase K followed by incubation with K63^2–7 (a) or K48^2–7 (b) poly-Ub chains at 4 °C for 4 h. c Confocal microscopy analysis for colocalization of pSC301-Mtb stably expressing green fluorescent protein (GFP) with K63^2–7 or K48^2–7 Ub chains in vitro. Bacteria (green) treated with (+) or without (–) proteinase K were further incubated with poly-Ub chains as in a and b, and were then immunostained using anti-Ub antibody (red). Scale bars, 10 μm. The percent colocalizations of Ub and Mtb are shown at the bottom. A total of 100 bacterial cells were counted. Two-tailed unpaired t-test was performed. d Schematic representation of the Mtb Rv1468c constructs used. e In vitro interaction analysis of Ub and GST-tagged full-length Mtb Rv1468c or the truncated mutants as indicated in d. Purified recombinant GST-tagged Mtb Rv1468c variants were immobilized on glutathione sepharose resin. Mono-Ub (His₆-tagged), K63 or K48-linked poly-Ub (Ub^2–7) was added, and then the protein complexes were assayed by immunoblotting with indicated antibodies. f Immunoblot analysis of proteins immunoprecipitated (IP) with anti-Flag M2 Affinity Gel from lysates of HEK293T cells transfected with empty vector (–) or hemagglutinin (HA)-tagged Ub (+) and Flag-tagged wild-type (WT) Rv1468c or its mutants (V64G or L65G). g Confocal microscopy analysis of HeLa cells cotransfected with vectors encoding HA-tagged Ub (red) and GFP-tagged WT Rv1468c, V64G or L65G mutant (green). Nuclei (blue) were stained with the DNA-binding dye DAPI. Scale bars, 10 μm. Results are representatives from at least three independent experiments (mean ± s.e.m. of n = 5 in c). The source data used in c are provided in Source Data