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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Leukemia. 2017 Oct 3;32(4):960–970. doi: 10.1038/leu.2017.304

Figure 5. BTK signaling can modulate phosphorylation of the 727 serine residue of STAT3.

Figure 5

Figure 5

(a) PBMCs from treatment-naïve CLL patients were cultured either alone or with ibrutinib (1 μM) for 24 hours and then stimulated with either CD40L (100 ng/ml) (left panel) or goat anti-human IgM+IgG (20μg/ml) (right panel) for 20 minutes. The cells were then fixed/permeabilized and stained for p-S727-STAT3 as described in the materials and methods section (n=5). Representative Phosflow histograms depicting p-S727-STAT3 expression on gated CD19+CD5+ CLL cells are shown. (b) Constitutive levels of p-S727-STAT3 in a BTK knockout RCH-ACV cells in comparison to RCH-ACV cells transfected with an empty vector. Cells were stained for viability using Live/Dead aqua, then fixed/permeabilized and stained for p-S727-STAT3 (n=4 BTK knockout, n=3 controls). Representative FACS plots showing p-S727-STAT3 reduction after BTK knockout are presented. The white histogram represents the isotype control while the light gray histogram shows p-S727-STAT3 expression in control RCH-ACV cells and the dark grey histogram shows p-S727-STAT3 downregulation in BTK knockout RCH-ACV cells. (c) BTK knockout and mock vector transfected RCH-ACV cells were stimulated with CD40L (100ng/ml) for 20 minutes and then stained for p-S727-STAT3 (n=4 BTK knockout, n=3 controls).