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Extended Data Fig. 4 | K11-linked ubiquitination of Rv0222 by ANAPC
suppresses TR AF6 signalling. a, b, Luciferase assay of HEK293T cells
transfected with a plasmid encoding NF-κB (a) or AP-1(b) luciferase reporter or
the indicated plasmids for 24 h. c, Immunoblots and immunoprecipitates of
HEK293T cell lysates transfected with the indicated plasmids. Six-week-old
female SCID mice were aerosol-infected with roughly 100 CFUs per mouse
of H37Rv, H37Rv(ΔRv0222 + GFP), H37Rv(ΔRv0222 + Rv0222) or
H37Rv(ΔRv0222 + Rv0222(K76A)). d, e, qPCR analysis of Il1b (d) and Il6 (e)
mRNA in lung tissues from mice infected for 7 days. f–h, Immunoblots and
immunoprecipitates of HEK293T cell lysates transfected with indicated
plasmids. i, Luciferase assay of control or ANAPC2-knockdown HEK293T cells
transfected with a plasmid encoding NF-κB luciferase reporter or the indicated
plasmids for 24 h. j–l, Immunoblots and immunoprecipitates of lysates from
control or ANAPC2-knockdown HEK293T cells transfected with the indicated
plasmids. m–o, qPCR analysis of Il1b (m), Il6 (n) and Il12 p40 (o) mRNA from
control or ANAPC2-knockdown THP1 cells infected with H37Rv or ΔRv0222 for
the indicated times (MOI = 5). p, Immunoblot of lysates from control or
ANAPC2-knockdown iBMDMs infected with H37Rv for the indicated times
(MOI = 5). Two-tailed unpaired Student’s t-test (a, b, d, e, i, m–o) were used for
statistical analysis. Data are representative of one experiment with at least
three independent biological replicates (c, f–h, j–l, p) and are mean ± s.e.m.
in a, b, i, m–o. d, e, Cumulative data from two independent experiments
(n = 6 mice). For gel source data, see Supplementary Fig. 1.