Neither proteasome inhibitors nor lysosomal inhibitors affect the levels of ubiquitinated Syk. (A) Washed platelets were pretreated with the proteasomal inhibitor epoxomicin (1 μM 30 minutes at 37°C) prior to activation by convulxin (100 ng/mL) for indicated times. The platelets were lysed with RIPA buffer and Syk was immunoprecipitated. The blot was probed for Syk. (B) Epoxomicin and MG262 inhibit platelet proteasomal enzymes. Washed platelets were incubated with either epoxomicin (1 μM) or MG262 (1 μM) for 30 minutes at 37°C. The platelets were washed and lysed as described. The lysates were assayed for proteasomal activity with Suc-Leu-Leu-Val-Tyr-AMC as substrate. (C) Measurement of the effect of epoxomicin (○) on ubiquitinated Syk using a phosphospecific (pY525/pY526) antibody. The experiment was similar to panel A, except the Syk immunoblot was probed with phosphospecific (pY525/pY526) antibody. The total material in the Syk and ubiquitinated Syk bands was quantitated for each incubation time and the area plotted in the figure. ▪ indicates control. The inset shows the 2 blots used. The data are representative of 3 experiments. (D) Washed platelets were pretreated with chloroquine (200 μM for 60 minutes at 37°C), a lysosomal inhibitor, prior to activation by collagen for indicated times. The platelets were lysed and Syk was immunoprecipitated. The blots were probed for Syk.