Pim kinases are involved in modulating SOCS-1 protein levels and SOCS-1 phosphorylation. (A) SOCS-1 protein levels are significantly reduced in Pim-deficient v-Abl–transformed pre-B cells. The expression of SOCS-1 protein was examined in 3 wild-type and 4 Pim-1^−/−/Pim-2^−/−/Pim-3^−/− (TKO) cell lines. Shown is a Western blot probed with indicated antibodies. (B) SOCS-1 levels in panel A were quantitated by densitometry and normalized to v-Abl expression levels. In each experiment, the highest level of SOCS-1 is 100. Plotted are the average levels of SOCS-1 from 3 independent experiments. Error bars represent SEM. (C) Various concentrations of the proteasome inhibitor LLNL were added to the cells, and cells were then harvested after 14 hours of incubation. The levels of indicated proteins were examined by Western blot. (D) v-Abl–transformed wild-type or TKO pre-B cells were exposed to cycloheximide (CHX). Cells were then harvested at the indicated time points and analyzed by Western blotting. (E) Blots in panel D were quantitated as described in panel B. Plotted are the results from 3 independent experiments. Error bars represent SD. (F) v-Abl–transformed wild-type or TKO cells were metabolically labeled with ³²PO₄. ³²P-labeled SOCS-1 was then isolated by immunoaffinity, and phosphorylation was detected by autoradiography. SOCS-1 expression levels were examined by Western blot. Lane 1, wild-type cells; lane 2, TKO cells.