Figure 4.
Figure 4. Effect of U73122, dimethyl BAPTA, and GF109203X on the tyrosine phosphorylation of PKCδ. Aspirin-treated and washed human platelets were stimulated with SFLLRN or AYPGKF in the presence or absence of U73122 (10 μM), dimethyl BAPTA (20 μM) (A) or GF109203X (1 μM) (B) at 37°C, and the reaction was stopped by adding 2 × cell lysis buffer. DMSO was used as a vehicle control. Stimulation times for SFLLRN and AYPGKF were 20 and 60 seconds, respectively. Incubation times for U73122, dimethyl BAPTA, and GF109203X were 10, 10, and 5 minutes at 37°C, respectively. PKCδ was immunoprecipitated as described, and the samples were analyzed for tyrosine phosphorylation by Western blotting using the monoclonal phosphotyrosine (4G10) antibody. Equal lane loading was assured by probing the samples with PKCδ antibody. The Western blot shown is representative of experiments done using platelets from 3 different donors.

Effect of U73122, dimethyl BAPTA, and GF109203X on the tyrosine phosphorylation of PKCδ. Aspirin-treated and washed human platelets were stimulated with SFLLRN or AYPGKF in the presence or absence of U73122 (10 μM), dimethyl BAPTA (20 μM) (A) or GF109203X (1 μM) (B) at 37°C, and the reaction was stopped by adding 2 × cell lysis buffer. DMSO was used as a vehicle control. Stimulation times for SFLLRN and AYPGKF were 20 and 60 seconds, respectively. Incubation times for U73122, dimethyl BAPTA, and GF109203X were 10, 10, and 5 minutes at 37°C, respectively. PKCδ was immunoprecipitated as described, and the samples were analyzed for tyrosine phosphorylation by Western blotting using the monoclonal phosphotyrosine (4G10) antibody. Equal lane loading was assured by probing the samples with PKCδ antibody. The Western blot shown is representative of experiments done using platelets from 3 different donors.

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