A C-terminal ER-targeting sequence promotes formation of an immature FLT3 receptor. (A) C-terminal sequence that confers FLT3 ER retention (designated R3), and corresponding control sequence that allows normal maturation, designated A3, are depicted. (B) HEK293 cells were transiently transfected with expression constructs for the indicated FLT3 versions and cell lysates were either directly or after FLT3 immunoprecipitation subjected to immunoblotting. R3 indicates FLT3 wild-type with the C-terminal ER retention sequence; A3, FLT3 wild-type with the corresponding control sequence, constructs in vector pAL; FLT3 WT, FLT3-ITD, constructs in pcDNA3. KA indicates the kinase-inactive K644A mutant. A vertical line has been inserted to indicate a repositioned gel lane. (C) 32D cells were stably transfected with FLT3 R3 or FLT3 A3. Cell extracts were subjected to immunoprecipitation, and the precipitates were digested with Endoglycosidase H (EndoH) or subjected to a corresponding mock treatment, as indicated, before sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. CG indicates complex glycosylated; HM, high-mannose; and DG, deglycosylated forms.