Human Flt-3/Flk-2 Antibody

Detection of Human Flt-3/Flk-2 by Western Blot

FLT3-ITD retention in the Golgi region is dependent on its tyrosine kinase activity. (a, b) MOLM-14 cells were treated for 4 h with AC220 (a) or PKC412 (b). Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. Full length blots are presented in Supplementary Fig. 5. (c) MOLM-14 cells were treated with AC220 (upper graph) or PKC412 (lower graph) for 48 h. Cell proliferation was assessed by ATP production. Results are means ± s.d. (n = 3). (d) Lysates from MOLM-14 were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H) then immunoblotted with anti-FLT3 antibody. CG complex-glycosylated form, HM high mannose form, DG deglycosylated form. Full length blots are presented in Supplementary Fig. 5. (e, f) MOLM-14 cells were treated with 10 nM AC220 or 100 nM PKC412 for 8 h (e) or 16 h (f). (e) Fixed cells were permeabilized, then immunostained with anti-FLT3 (red) and anti-calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (f) Non-permeabilized cells were immunostained with an anti-FLT3 extracellular domain (ECD) antibody. Bars, 10 µm. Note that FLT3 tyrosine kinase inhibitors inactivated FLT3, then released the receptor from the Golgi region for localization to the PM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

FLT3-ITD retention in the Golgi region is dependent on its tyrosine kinase activity. (a, b) MOLM-14 cells were treated for 4 h with AC220 (a) or PKC412 (b). Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. Full length blots are presented in Supplementary Fig. 5. (c) MOLM-14 cells were treated with AC220 (upper graph) or PKC412 (lower graph) for 48 h. Cell proliferation was assessed by ATP production. Results are means ± s.d. (n = 3). (d) Lysates from MOLM-14 were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H) then immunoblotted with anti-FLT3 antibody. CG complex-glycosylated form, HM high mannose form, DG deglycosylated form. Full length blots are presented in Supplementary Fig. 5. (e, f) MOLM-14 cells were treated with 10 nM AC220 or 100 nM PKC412 for 8 h (e) or 16 h (f). (e) Fixed cells were permeabilized, then immunostained with anti-FLT3 (red) and anti-calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (f) Non-permeabilized cells were immunostained with an anti-FLT3 extracellular domain (ECD) antibody. Bars, 10 µm. Note that FLT3 tyrosine kinase inhibitors inactivated FLT3, then released the receptor from the Golgi region for localization to the PM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

In AML cells, FLT3-ITD can activate AKT, ERK, and STAT5 before it reaches the PM. (a–c) MOLM-14 (a, b), MV4-11, or Kasumi-6 cells (c) were treated with inhibitors of ER export (BFA or M-COPA) for 8 h. (a) MOLM-14 cells treated with 1 µM BFA (middle panels) or 1 µM M-COPA (bottom panels) were stained with anti-FLT3 (red) and calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (b) Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. (c) MV4-11 (left) or Kasumi-6 cells (right) were treated with M-COPA for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. Note that BFA and M-COPA inhibited the activation of AKT and ERK but not that of STAT5 through blocking FLT3-ITD trafficking from the ER to the Golgi apparatus. (d, e) MOLM-14 cells were treated with monensin (inhibitor of Golgi export) for 8 h. (d) Cells treated with 100 nM monensin were stained with anti-FLT3 (red) and lectin-HPA (Golgi marker, blue). Dashed line, cell border. Bars, 10 µm. (e) Lysates were immunoblotted with the indicated antibody. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. (f) MV4-11 (left) or Kasumi-6 cells (right) were treated with monensin for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 6. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Flt-3/Flk-2 by Immunocytochemistry/ Immunofluorescence

In AML cells, FLT3-ITD can activate AKT, ERK, and STAT5 before it reaches the PM. (a–c) MOLM-14 (a, b), MV4-11, or Kasumi-6 cells (c) were treated with inhibitors of ER export (BFA or M-COPA) for 8 h. (a) MOLM-14 cells treated with 1 µM BFA (middle panels) or 1 µM M-COPA (bottom panels) were stained with anti-FLT3 (red) and calnexin (ER marker, green). Insets show the magnified images of the boxed area. Bars, 10 µm. (b) Lysates were immunoblotted for FLT3, phospho-FLT3 Tyr842 (pFLT3Y842), AKT, pAKT, ERK, pERK, STAT5, and pSTAT5. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. (c) MV4-11 (left) or Kasumi-6 cells (right) were treated with M-COPA for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. Note that BFA and M-COPA inhibited the activation of AKT and ERK but not that of STAT5 through blocking FLT3-ITD trafficking from the ER to the Golgi apparatus. (d, e) MOLM-14 cells were treated with monensin (inhibitor of Golgi export) for 8 h. (d) Cells treated with 100 nM monensin were stained with anti-FLT3 (red) and lectin-HPA (Golgi marker, blue). Dashed line, cell border. Bars, 10 µm. (e) Lysates were immunoblotted with the indicated antibody. To examine pFLT3Y591, FLT3 was immunoprecipitated, then immunoblotted. Full length blots are presented in Supplementary Fig. 5. (f) MV4-11 (left) or Kasumi-6 cells (right) were treated with monensin for 8 h, then immunoblotted. Full length blots are presented in Supplementary Fig. 6. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34811450), licensed under a CC-BY license. Not internally tested by R&D Systems.