Fig. 6
sCD4 disrupted MHCII/TLR4 rafts and reduced LPS/TLR4 inflammatory membrane confinement. Duolink assays to quantify protein-protein interactions of (a) the indicated pairs (red), or (b) between STING and SHP2 (green) that combined with immunofluorescent staining of MHC II (red) in BMDM. Tripartite colocalization indicated in yellow. The nuclei counter-stained with DAPI. Pearson’s coefficients indicated the degree of colocalization. Bar = 5 μm. c TNF and IL-6 in supernatants 30 min after peritoneal macrophages treated with LPS or LPS plus sCD4, in the presence of indicated endocytosis inhibitors. The average of two independent repeats. Three replicate wells were used for each condition where the indicated inhibitor was added. d Duolink spots of MHC II-SHP2 interactions and (e) flow cytometric analyses of cell surface MHC II. Bar = 5 μm. Macrophages (5 x 106) were treated with LPS/sCD4 for 1 h, and (f) endosomes were isolated for immunoblot analysis of the indicated proteins (asterisk), or (g) organelle numbers per cell were quantified after immunofluorescence staining of EEA1 (early endosomes), LAMP1 (lysosomes), RAB4 (recycling endosome) and RCAS1 (Golgi). h Lysosomes size was quantified using LysoTracker after RAW264.7 cells were transfected with GFP-tagged MHC II Aβ or MHC II AβΔCT. Several view fields were randomly selected and images were acquired every 10 s for 20 min of LPS or LPS plus sCD4 treatment. Mean ± SD are shown; n = 3–4 mice used where indicated; Statistics (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001;): Unpaired t test
