FIG. 3.

Characterization of the MHC-I-loaded perinuclear endosomal compartment in Δm138-MCMV-infected cells. (A) Colocalization of internalized MHC-I with early and late endosomal markers. At 4 h p.i., MHC-I molecules were labeled with MAbs and internalized for an additional 4 h. Internalized MHC-I-MAb complexes were stained with Alexa⁵⁵⁵-anti-mouse IgG2b secondary reagent (red fluorescence [a to d]) and colocalized with the early endosomal markers EEA1 (Alexa⁴⁸⁸-anti-chicken IgG [green fluorescence]) (a) and TfR (Alexa⁴⁸⁸-anti-rat IgG [green fluorescence]) (b) and the late endosomal markers Lamp1 (Alexa⁴⁸⁸-conjugated anti-rat IgG [green fluorescence]) (c) and LBPA (Alexa⁴⁸⁸-conjugated anti-mouse IgG1 [green fluorescence]) (d). Also shown is the colocalization of internalized MHC-I complexes (stained with Alexa⁴⁸⁸-anti-mouse IgG2b [green fluorescence]) with GM1 (stained with Alexa⁵⁵⁵-CTxB [red fluorescence]) (e) and fluid-phase internalized LysoTracker Red DND-99-labeled endosomes (red fluorescence) (f). (B) Internalized MHC-I-MAb complexes (red fluorescence) were colocalized with the Golgi marker GM130 (green fluorescence) in untreated cells or in cells treated for an additional 60 min with nocodazole (4 μM) and BFA (10 μg/ml). Bars, 10 μm.