Fig. 1.

Effect of H₂O₂and antioxidant treatment on autophosphorylation of FLT3 in intact cells and in vitro (A, B) RS4-11 cells were serum-starved and treated with H₂O₂ at the indicated concentrations at 37 °C for 30 min. Positive control was FL stimulation with 100 ng/ml for 10 min. Endogenous FLT3 was immunoprecipitated and autophosphorylation was detected with anti-pY591 FLT3 antibodies (denoted pFLT3); blots were stripped and reprobed with pan-specific anti-FLT3 antibodies (FLT3, C20). (A) Example experiment, (B) quantification of pY-FLT3/FLT3 ratios for multiple experiments (n = 5, significance tested with one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001 for comparison with control) (C, D) FLAG-tagged FLT3 was transiently expressed in HEK293 cells and enriched by anti-FLAG immunoprecipitation and elution with FLAG peptide under anaerobic conditions. Samples were treated with H₂O₂ for 10 min, and autokinase reactions were performed in absence or presence of 5 μM ATP and 5 mM MnCl₂ as indicated. Samples were analyzed by SDS-PAGE and immunoblotting with anti-pY591 FLT3 antibodies, and blots were reprobed with anti-FLT3 (S18). (C) Representative experiment, (D) quantification of pY-FLT3/FLT3 ratios for multiple experiments (n = 7). Box plot illustrating medians within boxes from first quartile (25th percentile) to the third quartile (75th percentile) and whiskers ranging from the 10th to the 90th percentiles. Differences did not reach significance by testing with one-way ANOVA. (E) MV4-11 cells were treated for 8 h with diphenyleneiodonium (DPI, concentrations indicated) or N-acetylcysteine (NAC, 10 mM), or the FLT3 kinase inhibitor AC220 (20 nM). Receptor autophosphorylation, and activation of the downstream mediators of cell transformation STAT5 and ERK1/2 were detected by immunoblotting using phosphospecific antibodies (pFLT3, pSTAT5, pERK1/2) and reblotting with corresponding pan-specific antibodies (FLT3 (S18), STAT5, ERK1/2). (F) Quantification of 6 experiments (treatment time 6–12 h). Values were calculated as arbitrary units (a.u.) relative to untreated cells and are given as means ± SEM. *p < 0.05, **p < 0.01,***p < 0.001 by one-way ANOVA compared to control.