Figure 2.

Phosphorylation of SHP-1 and SHP-2. PBMCs (1–2 × 10⁷) from different probands (wildtype Q551 or variant R551) were stimulated with IL-4 or IL-13 for 10 min. Cells were suspended in lysis buffer [10 mM Tris (pH 7.8), 5 mM EDTA, 50 mM NaCl, 30 mM pyrophosphate, 50 mM sodium fluoride, 20 μM sodium orthovanadate, 1%Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A and 10 μg/ml leupeptin; 10⁸cells/ml buffer] and incubated for 60 mins at 4°C. Insoluble material was removed by centrifugation and equal amounts of cell lysates (BioRad protein assay, BioRad Laboratories) were incubated with 1 μg/ml of the appropriate antibodies, anti-SHP1 or anti-SHP-2, followed by western blotting and immunostaining with either p-Tyr or the respective control antibody. (A). SHP-1, 3= WT/IL-13; 2= WT/IL-4; 1= WT unstimulated. (stain 2 min); 6= R551/ IL-13; 5= R551/ IL-4; 4= R551 unstimulated. (stain 5 min); (B). SHP-2, 3= WT/ IL-13; 2= WT/ IL-4; 1= WT unstimulated; 6= R551/ IL-13; 5= R551/ IL-4; 4= R551 unstimulated. PBMC = Peripheral blood mononuclear cell; SHP =SH2 containing phosphatase; WT = wildtype.