Figure 3.

Activation of PLCγ₂S707Y by catalytically-inert BTK is not mediated by augmented PLC substrate availability. A, COS-7 cells were transfected as indicated with 2 μg/well of empty vector or 0.5 μg/well of vector encoding WT BTK (WT) or BTK variants K430R, E41K, or E41K/K430R. Twenty four hours after transfection, the cells were incubated for 18 h with myo-[2-³H]inositol. Then, the amounts of radioactively labeled PtdIns (PI), PtdInsP (PIP), and PtdInsP₂ (PIP₂) in the intact COS-7 cells were determined by TLC analysis as described under “Experimental procedures.” Inset, cells from one well each were lysed and subjected to SDS-PAGE. Subsequent immunoblotting was performed using an antibody reactive against BTK or antibody reactive against β-actin. B, COS-7 cells were transfected as indicated with 500 ng of empty vector (Co.) or increasing amounts (15, 50, 150, or 500 ng) of vector encoding WT PLCγ₁ (γ₁WT), PLCγ₁S729Y (γ₁S729Y), WT PLCγ₂ (γ₂WT), or PLCγ₂S707Y (γ₂S707Y). C, COS-7 cells were transfected as indicated with 50 ng/well of vectors as described in B, and 100 ng/well of vector encoding WT BTK (WT) or BTK variants K430R, E41K, or E41K/K430R. Analysis of inositol phosphate formation was done as in Fig. 1. D, cells from one well each functionally analyzed in C were washed with 0.2 ml of Dulbecco's PBS and then lysed by addition of 100 μl of SDS-PAGE sample preparation buffer. Aliquots of the samples were subjected to SDS-PAGE, and immunoblotting was performed using an antibody reactive against the c-Myc epitope present on WT PLCγ₁, WT PLCγ₂, PLCγ₁S729Y, and PLCγ₂S707Y, antibody reactive against BTK, or antibody reactive against β-actin. A–C show representative results from three independent experiments each as mean values of three technical replicates.