Fig. 2: YspE2 of Y. pestis ubiquitinates hGBP1 and leads to its proteasomal degradation.

a Schematic diagram of the gene loci encoding the NELs of Y. pestis (gene IDs are labeled according to the annotation of Y. pestis 91001 genome). b Sequence analysis of the most closely related NELs from different pathogenic bacteria. c, d Empty vector of p3×Flag-CMV and the plasmids expressing YspE1, YspE2 and their ligase catalytic mutants, as well as YP_3417, were transiently co-transfected into 293 T with pCMV-Myc-hGBP1, and hGBP1 was detected by immunoblotting analysis (c) or co-immunoprecipitated with anti-c-Myc beads (d), followed by immunoblotting analysis using different antibodies. e HeLa cells stably expressing GFP-hGBP1 were infected with Pa3606, Y. pestis 201 and the various mutants in the presence or absence of MG132 as indicated, and the infected cells were collected at different times and subjected to SDS-PAGE separation and immunoblotting. f HeLa cells stably expressing GFP-hGBP1 were infected with ΔyspE2 complemented with pBAD24-YspE1 or pBAD24-YspE2, ΔyscI complemented with ΔyscI-pBAD24-YscI, and the infected cells were analyzed as described in e. g pCMV-Myc-hGBP1 was co-transfected into 293 T cells with p3×Flag-CMV-YspE2 or p3×Flag-CMV-YspE2_C386A, and hGBP1 was immunoprecipitated and detected using antibodies specific for K63-linked Ub, K48-linked-Ub, hGBP1 and FLAG-tag, respectively. K48-linked and K63-linked ubiquitin chains were run on SDS-PAGE and served as positive controls for immunoblotting detection. The image represents results from two independent experiments. h–j represented the quantitative and statistical analysis results of c, f and e (-MG132 only), respectively, from three independent experiments. Image J was used to obtain the quantitative results and bars represent mean ± S.D. Two-way ANOVA was performed to analyze the significance of difference in protein levels (**p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).