Fig. 5

Macrophage QKI depletion in post-MRSA infection was mediated by the ubiquitin–proteasome system. A Protein level of QKI was determined by western blot after RAW264.7 cells were stimulated by MRSA for indicated time. One representative immunoblot is shown (left). Graph (right) are representative quantification of the band intensity for immunoblots from three independent experiments. B Immunoblot analysis of whole-cell lysates from Raw264.7 cells after treatment of inhibitor “MG132” and infection with Mu50 for indicated durations. One representative immunoblot is shown(left), Graph (right) are representative quantification of the band intensity for immunoblots from three independent experiments. C RAW264.7 cells were incubated with bacteria and ubiquitin inhibitor “MG132” for 4 h, followed by nuclear (N) and cytoplasmic protein (C) extraction. Then the lysates were subjected to immunoprecipitation using anti-Flag and immunoblotted with indicated antibodies. D Immunoblot analysis of immunoprecipitated HA-tagged ubiquitin transiently expressed in RAW264.7 cells and incubating with Mu50 for 4 h, Ub, ubiquitin. The whole-cell lysates were subjected to immunoprecipitation using anti-HA under denaturing conditions, and immunoblotted with indicated antibodies. (E) RAW264.7 cells were co-transfected with indicated plasmids. At 36 h post-transfection, cells were infected for 4 h and lysed, followed by immunoprecipitation using anti-flag under denaturing conditions, followed by immunoblotting with indicated antibodies. All the bars represented the mean of measurements from three independent experiments, and the error bars indicated ± SD. (A–E) are representative of three experiments, A, ***p < 0.001 (student’s t test). B, *p < 0.05, not significant (ns), one-way ANOVA with Tukey’s multiple comparisons test