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. 2017 Apr 21;7:1009. doi: 10.1038/s41598-017-01246-w

Figure 6.

Figure 6

TRAF6 facilitates resistance of MyD88 to degradation. (a) Basal MyD88 expression is lowered by TRAF6 deficiency. Expression of MyD88, TIRAP, TRAF6, and actin in Traf6+/+ MEFs and Traf6−/− MEFs was assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. The uncropped images are in Supplementary Fig. 23. (b) MyD88 speckles are downregulated by TRAF6 deficiency. Immunofluorescent staining of Traf6+/+ MEFs and Traf6−/− MEFs for MyD88 (green) and LAMP2 (red) was carried out. Cell nuclei were stained with Hoechst 33342. Images were captured by means of a confocal microscope. Scale bar: 10 μm. (c) Stably expressed MyD88-GyrB is downregulated by TRAF6 deficiency. Traf6+/+ MEFs and Traf6−/− MEFs were stably transfected with a plasmid encoding FLAG-MyD88-GyrB or with an empty plasmid. Expression of MyD88, TRAF6, and α-tubulin was assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. The uncropped images are in Supplementary Fig. 24. (d) Speckles of MyD88-GyrB are downregulated by TRAF6 deficiency. Traf6+/+ MEFs and Traf6−/− MEFs were stably transfected with the FLAG-MyD88-GyrB construct. Immunofluorescent staining for FLAG (green) and LAMP2 (red) was carried out, and cell nuclei were stained with Hoechst 33342. Images were acquired by means of a confocal microscope. Scale bar: 10 μm. (e) Basal MyD88 in TRAF6-deficient cells is restored by TRAF6. Traf6−/− MEFs were transiently transfected with increasing amounts of a plasmid encoding FLAG-tagged wild-type TRAF6 (TRAF6 WT) or enzymatically inactive TRAF6 (TRAF6 C70A) or with an empty plasmid (mock). Expression of MyD88, FLAG-TRAF6, and α-tubulin was assessed by immunoblotting. Values within parentheses represent the ratio determined by densitometric measurement of the bands. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown. The uncropped images are in Supplementary Fig. 25. (e) MyD88-GyrB expressed in TRAF6-deficient cells is restored by TRAF6. Traf6−/− MEFs stably expressing FLAG-MyD88-GyrB were transiently transfected with a plasmid encoding TRAF6 WT or TRAF6 C70A, or an empty plasmid (mock). Expression of FLAG-MyD88-GyrB, endogenous MyD88, TRAF6, and α-tubulin was assessed by immunoblotting. All the blots were obtained under the same experimental conditions, and the cropped images of the blots are shown.