Induction of apoptosis in CLLB cellsby SKI-606 or R428 is associated with reduction of Axlphosphorylation. (A) SKI-606 targets Axl and SFK phosphorylation. Primary CLL B cells were treated with SKI-606 at sublethal doses as indicated for 24 hours. Cell lysates were examined for levels of Axl and SFK (Tyr 416) phosphorylation by Western blot analysis. Actin was used as loading control. (B) R428 inhibits Axl phosphorylation. CLL B cells were treated with R428 at a suboptimal dose (based on apoptosis studies) of 1.0μM for 24 hours, and cell lysates were examined for the Axl and SFK phosphorylation by Western blot analysis with the use of specific antibodies. Actin was used as loading control. Patients with patients were indicated by assigning arbitrary numbers. (C) Axl inhibition reduces Mcl-1. R428-treated CLL B-cell lysates (P5-P7) used above (B) were analyzed for the expression of Mcl-1, XIAP, and Bcl-2 by Western blots with the use of specific antibodies. A consistent reduction of Mcl-1 was detected in these cell lysates; however; only a subtle, if at all, or no effects on the expression of XIAP or Bcl-2 was observed on Axl-inhibition. Actin was used as loading control. (D) Knockdown of Axl reduces Mcl-1. CLL B cells were transfected with scrambled siRNA (Sc-siRNA) as a control or Axl-specific siRNA, and cell lysates were analyzed after 48 hours of transfection for the detection of P-Axl and Mcl-1 in Western blot. Actin was used as loading control. Patients with CLL are indicated by arbitrary numbers.