SEMA4A antibody is internalized in a temperature-dependent manner and localizes to the lysosomal compartment. (A) Immunofluorescence microscopy in NCI-H929 cells demonstrating localization of SEMA4A (green) and lysosomal-associated membrane protein 1 (LAMP-1) (red) before and after 3 hours of incubation at 37°C. Below the photomicrographs are plots showing fluorescence intensity as a function of distance from a reference point and confirming colocalization of SEMA4A with LAMP-1 after incubation. (B) Flow cytometry of NCI-H929 cells exhibiting the dynamics of SEMA4A internalization, as indicated by loss of antibody from the cell surface over time when cultured at 37°C but not at 4°C. (C) Histogram of cells from panel B, at the beginning and end of the time course. Internalization of SEMA4A from the cell surface affects the entire cell population. IgG1, immunoglobulin G1.