Figure 1.

Bruton tyrosine kinase is overexpressed and constitutively active in 32D myeloid progenitor cells ectopically expressing erythropoietin receptor and JAK2-V617F kinase. (A) 32D JAK2-wildtype (WT) and JAK2-V617F (VF) cells were starved of serum for 4 h and the whole cell lysates were analyzed for expression of phosphorylated (P) Bruton tyrosine kinase (BTK) (pY223) or p65 (pS536) and total (T) proteins. Densitometric analysis of P/T-BTK and -p65 is shown (n=5). (B) 32D VF cells were cultured in the presence of dimethyl sulfoxide (DMSO) or 0.5 μM ruxolitinib (RUX) overnight and the whole cell lysates were analyzed for expression of P-BTK or -p65 and T proteins. Densitometric analysis of P/T-BTK and -p65 is shown (n=4). (C) 32D WT and VF cells were electroporated with control (pGL4) or BTK promoter construct (pGL4-BTK) and treated with DMSO or 0.5 mM RUX overnight and promoter assays were performed as described previously⁵ (n=3). (D) 32D VF cells were cultured in the presence of DMSO or 0.5 mM RUX overnight and whole cell lysates were analyzed for expression of BTK. Densitometric analysis of T-BTK relative to vinculin is shown (n=4). Vinculin served as a loading control in all the experiments. Columns represent mean ± standard error of mean from independent experiments. Statistical significance between different conditions was calculated using the Student t-test; *P<0.05, **P<0.01 and ***P<0.001.