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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: J Immunol. 2013 Dec 11;192(2):714–721. doi: 10.4049/jimmunol.1302384

FIGURE 3. TRAF6 colocalizes with endosomal 2DL4.

FIGURE 3

A, The endocytosed 7A 2DL4 mutant colocalizes with wt 2DL4-gfp in HEK293T cells. Intact cells were incubated with anti-HA Alexa Fluor 594 at 37°C for 2 h, fixed and analyzed by confocal microscopy. The arrow in the third panel indicates the axis along which the fluorescence intensities shown in the fourth panel were traced. B, An intact TRAF6-binding domain is required for co-localization with endosomal 2DL4. HEK293T cells were mock-transfected (bottom panel) or transiently transfected with plasmids encoding either wt 2DL4 (top panel) or 7A 2DL4 (middle panel). Intact cells were incubated with anti-HA Alexa Fluor 488, fixed, permeabilized, stained with anti-TRAF6 antibody plus secondary antibody conjugated with Alexa Fluor 564, and analyzed by confocal microscopy. wt and 7A 2DL4 are shown in green, and endogenous TRAF6 is shown in red. Scale bar: 20 μM C, Pearson's Correlation Coefficient for co-localization of endogenous TRAF6 with wt or 7A 2DL4 in the transfected HEK293T cells. Results are presented as mean ± SEM of 12 cells transfected with WT 2DL4 and 10 cells transfected with 7A 2DL4 (*P<0.0001; Student's t-test).