Al fluorescence microscopy. (B) YOD1 and TRAF6 co-localize in cytosolic speckles
Al fluorescence microscopy. (B) YOD1 and TRAF6 co-localize in cytosolic PTPRC/CD45RA Protein supplier speckles upon co-expression. The co-localization is independent of YOD1 catalytic activity. GFP-YOD1 (WT or C160S) and RFPTRAF6 had been co-transfected in U2OS cells and localization was analyzed as in (A). Enlargement of boxed region is shown next to Merge. Plot Profile analysis was performed to quantify fluorescence intensities and to monitor co-localization along the white line. (C) TRAF6 interacts with YOD1 independent of its catalytic activity. HEK293 cells have been co-transfected with HA-TRAF6, GFP-YOD1 WT and GFP-YOD1 C160S constructs as indicated. Co-IP was carried out working with anti-HA antibodies and analyzed by Western Blot. Merged photographs include things like nuclear staining with Hoechst 33342. Scale bars depict 10 mM (A and B). DOI: 10.7554/eLife.22416.006 The following figure supplement is accessible for figure 2: Figure supplement 1. YOD1/TRAF6 co-localization. DOI: ten.7554/eLife.22416.The staining and localization with the YOD1/TRAF6 speckles displayed similarities to cytoplasmic aggregates termed sequestosomes that have been described for the TRAF6 interactor p62/Sequestosome-1 (Sanz et al., 2000; Seibenhener et al., 2004; Wang et al., 2006). As anticipated, we observed related aggregates when Crimson-p62 was expressed in U2OS cells (Figure 3A) and TRAF6 was recruited to the punctuated p62 sequestosomes in U2OS cells as evident from plotted FI of CFP-p62 and RFP-TRAF6 spots at the same time as co-clustering of FI as measured by automated image evaluation (Figure 3B and Figure 3–figure supplement 1A). In contrast, GFP-YOD1 was not co-localizing with Crimson-p62 aggregates. Having said that, it appeared that p62 staining was slightly a lot more diffuse in YOD1 expressing cells, hinting at an indirect effect of YOD1 around the formation of p62 sequestosomes (Figure 3B and Figure 3–figure supplement 1B). We confirmed by co-IP that p62 binds to FLAG-TRAF6, but not to FLAG-YOD1 in HEK293 cells (Figure 3–figure supplement 1C). The MATH domain of TRAF6 serves as interaction and oligomerization platform and therefore it is actually critically involved in regulating TRAF6 functions in NF-kB signaling (Ye et al., 2002; Walsh et al., 2015).Schimmack et al. eLife 2017;6:e22416. DOI: ten.7554/eLife.5 ofResearch articleCell BiologyACrimson-pRFP-TRAFBRFP-TRAF6 CFP-p62 MergeCrimson/CFP-p62 GFP-YODGFP-YODCrimson-pMergePlot! CHA-TRAF6 356-501 HA-TRAF6 310-522 HA-TRAF6 287-501 Crimson-p62 GFP-YODF+ + + + + + + + + + + + + + + +GFP-YOD1 WTGFP-YOD1 C160SRFP-TRAFYOD1 pRFP-TRAFHA-IPHACrimson-pCrimson-p55YOD1 pLysates25MergeHAMergeDMYC-TRAF6 Crimson-p62 GFP-YOD1 C160S GFP-YOD1 WT + + + + + + + + + p62 MYC-IP + + +Ens nsYOD1 TRAF TRAF6 YOD1 p62 TRAF6 YOD1 C/S p62 GFP-YOD1 RFP-TRAF6 Crimson-pp62 YOD1 Lysates TRAF6 GAPDHFigure three. YOD1 competes with p62 for Delta-like 1/DLL1 Protein Formulation binding to TRAF6 and recruitment to sequestosomes. (A) p62 localizes to sequestosomes. Crimson-p62 was transfected in U2OS cells and localization was analyzed by confocal fluorescence microscopy. (B) TRAF6, but not YOD1, is recruited to p62-containing aggregates. RFP-TRAF6 and CFP-p62 or GFP-YOD1 and Crimson-p62 were co-transfected in U2OS cells and localization was analyzed as in (A). Enlargement of boxed region is shown subsequent to Merge. Plot Profile evaluation was conducted to quantify fluorescence intensities and monitor co-localization Figure three continued on next pageSchimmack et al. eLife 2017;six:e22416. DOI: 10.7554/eLife.6 ofResearch article Figure 3 continuedCell Biologyalong the white line. (C) Y.