Eμ-Tcl1 CLL cells have constitutively activated BCR signaling that regulates Par-4 expression. (A) Western blot of Eμ-Tcl1 CLL cells treated with 5 μM fostamatinib for different time points. (B) Western blot of Eμ-Tcl1 CLL cells treated with 5 μM ibrutinib. (C) Survival of Eμ-Tcl1 CLL cells, 2 human CLL (hCLL) cell lines (Mec-1, OSU-CLL), and primary human CLL cells is dependent on BCR signaling. Cells were treated with different concentrations of dasatinib for 48 hours and viability was measured by MTT assay in triplicates. (D) Primary Eμ-Tcl1 CLL cells (left) or Mec-1 CLL cells (right) were treated with 1 μM dasatinib and the protein lysates were analyzed by western blot. (E) Treatment of Eμ-Tcl1 CLL cells (left) or Mec-1 cells (right) with dasatinib decreases Par-4 mRNA as measured by qRT-PCR. Par-4 mRNA is normalized to mouse 18S mRNA expression. In immunoblots, phosphorylated protein values were normalized to total protein. β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize total protein levels (Lyn, Syk, Btk, and Par-4) in the samples. Results are representative of 3 or more experiments.