The target of AT1413 is a novel sialylated CD43 epitope (CD43s). (A) IP with biotin-labeled, sortase-tagged AT1413 of THP-1 or Jurkat lysate. Imperial Coomassie-stained gel. (B) Western blot analysis of the AT1413 and AT1002 immunoprecipitates of THP-1 and Molm13 lysates with Mem59 mouse-anti-human CD43 antibody. A vertical line has been inserted to indicate the repositioned marker lane. (C) Deglycosylation of THP-1 cells with neuraminidase (sialidase) abrogated binding of antibodies AT1413, Mem59, DF-T1, and 84-3C1 in a dose-dependent manner. Clone L10 does not target a sialylated epitope of CD43.36 (D) Staining of THP-1 and Jurkat cells with AT1413 and with the commercially available CD43-specific antibodies DF-T1, L10, and Mem59. (E) Competition experiment with AT1413 and commercially available CD43-specific antibodies. THP-1 cells were incubated with indicated antibodies, biotinylated AT1413 and streptavidin PECy7. AT1413 binding to THP-1 target cells was not affected by preincubation of the cells with commercially available CD43 antibodies, but was inhibited in a dose-dependent manner when THP-1 cells were preincubated with unlabeled AT1413. (F) Immunoprecipitation with AT1413 of truncated variants of THP-1 expressed FLAG-tagged CD43. Immunoblotting with FLAG antibody revealed binding of AT1413 to CD43 mutants A-F and no binding to mutants H-K. Truncations were performed as indicated in supplemental Figures 3 and 4. Ctr, control with GFP-transduced THP-1 cells. (G) AT1413 (2.5 µg/mL) binding of the human cell line THP-1 and the murine AML cell line WEHI-3b. (H) Western blot analysis of the AT1413 and AT1002 immunoprecipitates of the mouse AML cell line WEHI-3b lysate with anti-mouse CD43 antibody.
