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Time-lapse microscopy For videos of Sema4D-Fc-647 endocytosis, HeLa cells expressing GFP-tubulin and mCherry-Histone plated in chamber slides were incubated with Sema4D-Fc-Alexa647 in FluoroBrite DMEM Media (Thermo Fisher Scientific) with 10% serum, 1 mM sodium pyruvate and 2 mM glutamine and imaged using a Nikon Inverted spinning disk confocal microscope at 40

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