Figure 5.
MS4A3 traffics between plasma membrane and endosomes to regulate the endocytosis surface-bound protein. (A) Representative confocal microscopy images of LAMA-84 cells expressing MS4A3-EGFP fusion protein. The arrow depicts a typical intracellular vesicle with MS4A3-EGFP accumulation. Upper scale bar: 10 μm, lower scale bar: 5 μm. (B) Confocal microscopy-based colocalization analysis of MS4A3-EGFP with various intracellular structures in HEK293T cells. Left: extent of colocalization quantified as Pearson’s coefficient value. Right: relative fluorescent intensity of marker proteins in MS4A3-EGFP positive vesicles. (C) Confocal microscopy showing the accumulation of MS4A3-EGFP in late endosomes, and near lysosomes, in LAMA-84 cells. Boxes indicate areas of higher magnification. Color-coded arrows point to representative puncta of overlapping or adjacent signals. (D) Trafficking of cell surface MS4A3 (extracellularly labeled by monoclonal antibody, red) to endosomal MS4A3 pool (shown by MS4A3-EGFP, green puncta) after 5 minutes of incubation at 37°C. Arrows point at representative endosomes with internalized surface MS4A3 (red + green). (E) Quantification of cells containing internalized MS4A3 from experiment in panel D. (F) Streptavidin blotting (left) and Coomassie blue staining (right) of sulfo-NHS-biotin-labeled LAMA-84 cells with or without MS4A3 KD. (G) Tandem MS identified surface-bound proteins whose internalization is mediated by MS4A3. LAMA-84 cells with dox-shMS4A3 were cultured with or without 100 ng/mL dox for 72 hours before sulfo-NHS-SS-biotin labeling, streptavidin-bead pulldown, and MS analysis. (H) HEK293 cells were transfected with MS4A3-EGFP fusion and CSF2RB-RFP fusion protein plasmids, and cells were fixed and imaged by confocal microscopy. Arrows point at intracellular vesicles with overlapping green and red signals. (I) LAMA-84 cells were transfected with MS4A3-EGFP fusion and CSF2RB-RFP fusion protein plasmids by electroporation, and the colocalization between the 2 proteins were analyzed by FRET signals on a flow cytometer with multiple detection filters for each laser.

MS4A3 traffics between plasma membrane and endosomes to regulate the endocytosis surface-bound protein. (A) Representative confocal microscopy images of LAMA-84 cells expressing MS4A3-EGFP fusion protein. The arrow depicts a typical intracellular vesicle with MS4A3-EGFP accumulation. Upper scale bar: 10 μm, lower scale bar: 5 μm. (B) Confocal microscopy-based colocalization analysis of MS4A3-EGFP with various intracellular structures in HEK293T cells. Left: extent of colocalization quantified as Pearson’s coefficient value. Right: relative fluorescent intensity of marker proteins in MS4A3-EGFP positive vesicles. (C) Confocal microscopy showing the accumulation of MS4A3-EGFP in late endosomes, and near lysosomes, in LAMA-84 cells. Boxes indicate areas of higher magnification. Color-coded arrows point to representative puncta of overlapping or adjacent signals. (D) Trafficking of cell surface MS4A3 (extracellularly labeled by monoclonal antibody, red) to endosomal MS4A3 pool (shown by MS4A3-EGFP, green puncta) after 5 minutes of incubation at 37°C. Arrows point at representative endosomes with internalized surface MS4A3 (red + green). (E) Quantification of cells containing internalized MS4A3 from experiment in panel D. (F) Streptavidin blotting (left) and Coomassie blue staining (right) of sulfo-NHS-biotin-labeled LAMA-84 cells with or without MS4A3 KD. (G) Tandem MS identified surface-bound proteins whose internalization is mediated by MS4A3. LAMA-84 cells with dox-shMS4A3 were cultured with or without 100 ng/mL dox for 72 hours before sulfo-NHS-SS-biotin labeling, streptavidin-bead pulldown, and MS analysis. (H) HEK293 cells were transfected with MS4A3-EGFP fusion and CSF2RB-RFP fusion protein plasmids, and cells were fixed and imaged by confocal microscopy. Arrows point at intracellular vesicles with overlapping green and red signals. (I) LAMA-84 cells were transfected with MS4A3-EGFP fusion and CSF2RB-RFP fusion protein plasmids by electroporation, and the colocalization between the 2 proteins were analyzed by FRET signals on a flow cytometer with multiple detection filters for each laser.

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