Intracellular retention of ER-targeted FLT3. (A) 32D cells expressing FLT3 A3, or FLT3 R3, FLT3 D835Y, FLT3 D835Y R3, or FLT3 D835Y A3, FLT3 ITD, FLT3 ITD A3, or FLT3 ITD R3, as indicated, were subjected to FACS analysis using PE-labeled anti-FLT3 antibodies. The analysis was performed without or with prior cell permeabilization. Parental 32D cells were analyzed in parallel as negative controls for all series (red curves). An example analysis is shown (top left). (B) Cellular localization of FLT3 A3 or FLT3 R3, FLT3 D835Y R3, or FLT3 D835Y A3, FLT3 ITD, FLT3 ITD A3, or FLT3 ITD R3 (as indicated) shown by immunofluorescence using confocal laser scanning microscopy using equipment described in “Immunocytochemical analysis of FLT3 in 32D cells.” Counterstaining of the cells was performed with an antibody against the ER-resident protein calnexin or with fluorescently labeled wheat-germ agglutinin to mark the cell surface, as indicated. Scale bars correspond to 5 μm.