Fig. 6

Ubiquitination of QKI was mediated by RNF6. A Identification of differentially expressed proteins between flag-QKI –transfected (infection) and flag-QKI control (without infection)– transfected RAW264.7 cells. Negative controls with or without infection were internal controls respectively. LC–MS/MS, liquid chromatography coupled to tandem mass spectrometry. Volcano graph showed different expressions of protein after infection. The y-axis corresponds to the mean expression value of log10 (p-value), and the x-axis displays the log2 fold exchange value. The red dots represent the up regulated expressed transcripts [(p < 0.05) and fold change > 2]. The blue dots represent the transcripts whose expression down regulated [(p < 0.05) and fold change > 2]. Red and blue dots represent probe sets for protein expression at significantly higher (n = 70) or lower (n = 59) levels during infection. B Immunoblot analysis of expression of Rnf6 after RAW 264.7 cells infection for indicated durations, with actin as an internal control. One representative immunoblot is shown (on the left). Graphs (on the right) are representative quantification of the band intensity for immunoblots from three independent experiments. C RAW264.7 cells were infected with siRNA for Rnf6 (Si-1, Si-2, Si-3) and NC for control. Cell lysates were subjected to western blot to analyze the protein expression of Rnf6, with actin as an internal control. One representative immunoblot is shown (on the left). Graphs (on the right) are representative quantification of the band intensity for immunoblots from three independent experiments. D Immunoblot analysis of expression of QKI and Rnf6 after Raw264.7 was transfected with Myc-tagged Rnf6 vector, siRnf6, and NC vector with Mu50 infection or not. One representative immunoblot is shown (on the left). Graphs (on the right) are representative quantification of the band intensity for immunoblots from three independent experiments. E Immunoblot analysis of immunoprecipitated Myc-tagged Rnf6 transiently expressed in RAW264.7 cells. After infection of Mu50 for 4 h, the whole-cell lysates were subjected to immunoprecipitation using anti-Myc under denaturing conditions, and immunoblotted with indicated antibodies. F RAW264.7 cells were transfected with Myc-tagged Rnf6 vector, Flag-tagged QKI vector and siRNA of Rnf6 as indicated. After infection of Mu50 for 4 h, the whole-cell lysates were subjected to immunoprecipitation using anti-Flag under denaturing conditions, and immunoblotted with indicated antibodies. G–H Immunoblot analyzed the expression of PI3K-p110α, PI3K-p110βand LC3 in RAW264.7 cells after transfected with Myc-tagged Rnf6 and siRnf6. After infection of Mu50 for 4 h, the whole-cell lysates were subjected to immunoblot analysis using indicated antibodies. One representative immunoblot is shown. Graphs (Figure H) are representative quantification of the band intensity for immunoblots from three independent experiments. I RAW264.7 cells were transfected with siRNA of siRnf6, siQKI or both respectively, followed with phagocytosis assay. At 8 h post-incubation, the number of bacteria units forming was counted. Enumeration of forming bacteria units (c.f.u) were showed. All the bars represented the mean of measurements from three independent experiments, and the error bars indicated ± SD. (B–H) are representative of three experiments, *p < 0.05, **p < 0.01, ***p < 0.001, not significant (ns), one-way ANOVA with Tukey’s multiple comparisons test