Fig. 8.
The calcium-sensitive tyrosine kinasePYK2 is activated after βA andPrP fibril stimulation of THP-1 cells. THP-1 cells were incubated in DMSO vehicle (c), 50 nm thapsigargin, 10 μmDTBHQ, 5 μmPP1, or 2 μmGo6976 (45 min; 37°C for each) before incubation inPrP 106–126 (80 μm; 5 min) orβA 25–35 (40 μm; 2 min) or cross-linking of FcγRII [25 μg/ml anti-FcγRII Ab + 100 μg/ml GαM Fab2 (15 min on ice with Ab + 2 min at 37°C with Fab2)] or Con A (60 μg/ml; 5 min) as a positive control. The tyrosine kinase PYK2 and the cytoskeletal protein paxillin were immunoprecipitated from lysates of THP-1 cells and resolved by SDS-PAGE. The anti-phosphotyrosine antibody 4G10 was used to Western blot the immunoprecipitated proteins. The blots were stripped and reprobed with the immunoprecipitating antibodies to normalize for protein load. A, B,D, E, Changes in PYK2(A, B) and paxillin (D,E) tyrosine phosphorylation were observed after pretreatment of THP-1 cells with 10 μmDTBHQ, 5 μmPP1, or 2 μmGo6976 before stimulation withPrP 106–126 and βA 25–35.C, F, Changes in PYK2(C) and paxillin (F) tyrosine phosphorylation were also observed after pretreatment of THP-1 cells with 2 μmGo6976 before stimulation with 100 nmTPA.