PCI-32765 antagonizes BCR dependent signaling pathways and cell proliferation. (A) CD19⁺ cells from CLL patients (N = 12) were immunoprecipitated for 4G10. BTK phosphorylation at tyrosine sites was then evaluated by immunoblot analysis. Quantification was done using the Alpha Innotech FluorChemQ MultiImage III system. Dark line represents average. (B) CD19⁺ cells from CLL patients (N = 4) were treated with and without 10μM PCI-32765 for 2 hours. Cells were immunoprecipitated with 4G10 phospho-tyrosine antibody. BTK phosphorylation at tyrosine sites was then evaluated by immunoblot analysis. Results are shown from 1 of 4 experiments. (C) CD19⁺ cells from CLL patients (N = 7) were incubated with various concentrations of PCI-32765 for 1 hour. ERK phosphorylation at Thr²⁰²/Tyr²⁰⁴ was assessed by immunoblot. Results are shown from 1 of 3 experiments. (D) CD19⁺ cells from CLL patients (N = 4) were incubated with 1 or 10μM PCI-32765 and/or 1 μg/mL CD40L for 1 hour. AKT phosphorylation at ser⁴⁷³ was assessed by immunoblot. Results are shown from 1 of 3 experiments. (E) CD19⁺ cells from CLL patients (N = 3) were incubated with 10μM PCI-32765 and/or 1 μg/mL CD40L for 1 hour. EMSA analysis was done with nuclear extract using a radio-labeled oligonucleotide containing a consensus NF-κB binding site. Antibody shifts were performed from CD40L treated sample incubated with antibodies specific to the p65 or p50 subunits of NF-κB. The p65/p50 complex is indicated. Results are shown from 1 of 3 experiments. (F) CD19⁺ cells from CLL patients (N = 7) were incubated with or without 10μM PCI-32765 and 3.2μM CpG685. Proliferation was assessed by tritiated thymidine incorporation.