Fig. 6. Bmx/Etk is the downstream target for PI3K upon GPR55 stimulation by anandamide. (A) Phosphorylation of Bmx/Etk upon stimulation with 10 µM anandamide in the nominal absence of extracellular Ca²⁺ was tested by immunoprecipitation using anti-Bmx/Etk antibody (upper blot) and subsequent staining with the anti-phosphotyrosine antibody 4G10 (lower blot). (B,C) The effects of the Tec-family (Bmx/Etk) kinase inhibitor LFM-A13 (10 µM, n=46; control, n=30) or pretreatment with siRNA against Bmx/Etk (n=6; control, n=27) on anandamide (10 µM) -induced cytosolic Ca²⁺ signaling were monitored in nominally Ca²⁺-free solution. (D) The effect of the Tec-family (Bmx/Etk) kinase inhibitor LFM-A13 (10 µM, n=75; control, n=71) on O1602 (10 µM) -trigged cytosolic Ca²⁺ signaling in Ca²⁺-containing buffer was tested. Standard immunoprecipitation was applied. In experiments with siRNA, cells were transiently transfected with a vector encoding approved siRNA against Bmx/Etk. Experiments were performed 48 hours after transfection. Cytosolic free-Ca²⁺ concentrations were recorded using fura-2. ^*P<0.0001 versus the absence of the inhibitor.