Journal: Virulence

Article Title: Macrophage innate immune responses delineate between defective translocon assemblies produced by Yersinia pseudotuberculosis YopD mutants.

doi: 10.1080/21505594.2023.2249790

Figure Lengend Snippet: Figure 2. YopD variants control Y. pseudotuberculosis mediated suppression of p38 MAPK. RAW 264.7 macrophages were infected with parental Y. pseudotuberculosis or the ΔyopJ mutant strain at a MOI of 20 (a). At different time points post-infection (15, 30, 45, and 60 min), cells were lysed and equal amount of protein were resolved on SDS-PAGE. Active p38 was detected by western blot using phospho-p38 antibodies. Total p38 and GAPDH were used as a loading control. The displayed western blot image is one experiment. The scatter plot beneath this image represents the quantification achieved from four independent experiments (n = 4), and illustrates the time-dependent suppression of phospho-p38 by infection with parental bacteria compared to infection with the ΔyopJ mutant. In a subsequent series of experiments using the same conditions, RAW 264.7 macrophages were infected with parental Y. pseudotuberculosis, the ΔyopJ mutant strain, or strains producing variants of YopD (b). At two pre-selected time points post-infection (15 and 60 min), cells were lysed and equal amount of protein were resolved on SDS-PAGE. Active p38 was detected by western blot using phospho-p38 antibodies. Total p38 and GAPDH were used as a loading controls. The displayed western blot image is one experiment. The scatter dot plot beneath this image represents the quantification achieved from four independent experiments (n = 4), and illustrates the time-dependent suppression of phospho-p38 by infection with parental bacteria compared to infection with bacteria producing the various YopD mutants. In all cases, quantification of western blots was performed using ImageJ software. The phospho-p38 data was normalized against total p38. Significance was determined from biological replicates using a two-tailed, unpaired Student’s t-test. The double asterisk (**) and single asterisk (*) reflects the degree of significant difference with p < 0.01 and p < 0.05, respectively.

Article Snippet: Specific membrane-bound proteins were detected with anti-phospho-p38 MAPK (Thr180/Tyr182, #4511) (D3F9) HyCloneTM, anti-total p38 MAPK (#9212) Rabbit mAb (Cell Signaling Technology Europe, B.V., Leiden, The Netherlands) or mouse derived anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, clone 6C5 (#MAB374, Merck KGaA, Darmstadt, Germany).

Techniques: Control, Infection, Mutagenesis, SDS Page, Western Blot, Bacteria, Software, Two Tailed Test

Journal: Virulence

Article Title: Macrophage innate immune responses delineate between defective translocon assemblies produced by Yersinia pseudotuberculosis YopD mutants.

doi: 10.1080/21505594.2023.2249790

Figure Lengend Snippet: Figure 3. Y. pseudotuberculosis-mediated inhibition of p38 and NF-κB is influenced by the function of YopD. (a-b) RAW 264.7 cells were infected with parental Y. pseudotuberculosis or strains producing the variants of YopD at a MOI of 20. At 4 h post-infection, cells were fixed, permeabilized and incubated sequentially with anti phospho-p38 (a) or anti NF-κB (b) antibodies and then with AlexaFlour-488 or -568 conjugated secondary antibodies. Cells were counter-stained for nuclei with DAPI, and visualized by confocal microscopy. The dashed lines in the panel furtherest to the right in part (b) were used for calculation of Pearson coefficient for colocalization of NF-κB and DAPI. (c) The histogram to the left indicates quantification of phospho-p38 staining intensity of the uninfected and infected RAW 264.7 cells. (d) The histogram to the right indicates quantification of nuclear NF-κB staining intensity of the uninfected and infected RAW 264.7 cells. The quantification was carried out using ImageJ software. Data points indicate fluorescence intensity for randomly selected cells from two independent experiments (n = 50 cells). Histogram show mean ± S.D. Significance was determined from replicates using a one-way analysis of variance (ANOVA) with Dunnett’s post-test against cells infected with parent strain of Y. pseudotuberculosis. ****p < 0.0001, *p < 0.05. Or ns = not significant. Scale bars = 20 μm.

Article Snippet: Specific membrane-bound proteins were detected with anti-phospho-p38 MAPK (Thr180/Tyr182, #4511) (D3F9) HyCloneTM, anti-total p38 MAPK (#9212) Rabbit mAb (Cell Signaling Technology Europe, B.V., Leiden, The Netherlands) or mouse derived anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, clone 6C5 (#MAB374, Merck KGaA, Darmstadt, Germany).

Techniques: Inhibition, Infection, Incubation, Staining, Confocal Microscopy, Software, Fluorescence