Figure 4

NF-κB signaling increased during early Mk differentiation and decreased during terminal differentiation. (A) Cartoon depicting simplified NF-κB signaling pathway. 'P' denotes phosphorylated form of protein. (B) IκBα phosphorylation in Mk cytokine-cocktail-stimulated cultures was examined by flow cytometry. Cells were stained with anti-CD41a-FITC and rabbit anti-phospho-IκBα (Ser32) primary antibody followed by APC-conjugated goat anti-rabbit secondary antibody. Results are from one representative experiment (n = 2). (C) Kinetics of NF-κB p65 phosphorylation (Ser529) in CD41a⁺ cells from Mk cytokine-cocktail-stimulated cultures was measured by flow cytometry. Data shown is geometric mean fluorescence intensity of anti-phospho-p65-antibody stained samples relative to cells stained with isotype-matched control antibodies. Results are from one representative experiment (n = 2). (D) Q-RT-PCR analysis of IκBα mRNA expression in primary human CD34⁺ cells stimulated with 100 ng/mL Tpo.