Fig. 1

a CLL B-cells (N = 8) were plated in 96-well plates at 400,000 cells per well. Cells were treated with either vehicle, 0.5uM SNX-2112, or 3.2uM CpG + 0.5uM SNX-2112 for 48 h followed by addition of MTS reagents and samples were read at 490nm. b CLL B-cells (N = 8) were treated with either vehicle, 3.2uM CpG, or 3.2uM CpG + 0.5uM SNX-2112. CD19+ and live cells were stained and analyzed by flow cytometry for HLA-DR and CD86 surface expression. c CLL B-cells isolated from the peripheral blood of patient samples (N = 7) were treated with vehicle or 0.5uM SNX-2112 for 16 h. Whole cell lysates were isolated and immunoblots performed to determine total levels of BTK, AKT and Hsp70 protein, as well as the loading control GAPDH (left). Immunoblots for all patient samples were quantified and the fold change in protein is shown (right; normalized to GAPDH then displayed as fold change relative to the vehicle treatment). The red line indicated the average fold change for all 7 samples. The control lysate (Ctrl) is isolated from Mec1 B-cells. d XLA cell lines (BTK null) were transfected to express either wild type or C481S mutant BTK. Cell lines were then treated for 16 h with vehicle, 1uM ibrutinib (for 1 h followed by washout), or 100nM SNX-2112. Whole cell lysates were collected and immunoblot analysis performed for phospho-BTK and total BTK, as well as the loading control Actin. Immunoblots were quantified and the fold change in protein is shown below the graph (normalized to Actin then displayed as fold change relative to the vehicle). The control lysates are parental 293T cells (Ctrl -) and 293T cells over-expressing BTK protein (Ctrl +)