Different signaling quality of ER-bound and surface FLT3. (A) 32D cells expressing FLT3 R3 or FLT3 A3 were starved and stimulated with FL (100 ng/mL, 10 minutes) or left unstimulated, as indicated. FLT3 was immunoprecipitated and analyzed by immunoblotting with anti-FLT3 or anti-phosphotyrosine antibodies (4G10). (B) 32D cells expressing FLT3 R3, FLT3 A3, or FLT3 ITD, as well as parental 32D cells, were starved and stimulated with FL (100 ng/mL, 10 minutes) or left unstimulated, as indicated. Lysate aliquots were subjected to analysis of phosphorylated signaling proteins and of the corresponding proteins for loading controls, as indicated. (C) 32D cells expressing FLT3 D835Y R3 or FLT3 D835Y A3 were starved and stimulated with FL (100 ng/mL, 10 minutes) or left unstimulated, as indicated. FLT3 was immunoprecipitated and analyzed by immunoblotting with anti-FLT3 or anti-phosphotyrosine antibodies (4G10). (D) Signal transduction of FLT3 D835Y R3– and FLT3 D835Y A3–expressing cells was analyzed as described in panel B. (E) 32D cells expressing FLT3 ITD, FLT3 ITD R3, or FLT3 ITD A3 were starved, and FLT3 was immunoprecipitated and analyzed by immunoblotting with anti-FLT3 or anti-phosphotyrosine antibodies (4G10). (F) Constitutive signal transduction of FLT3 D835Y ITD, FLT3 ITD A3, and FLT3 ITD R3 was analyzed by immunoblotting in lysates of starved cells.