Figure 6. Effects of pVHL on BIM_EL polyubiquitination and its stability after serum-stimulation.

(A) COS-7 cells were transiently transfected with 0.2 μg of BIM_EL plasmid with or without 1 μg of pVHL plasmid. The next day, whole cell lysates were prepared and immunoblotted for BIM_EL, pVHL and α-tubulin. Note that co-expressing pVHL results in an increase in the levels of BIM_EL under these conditions. (B) COS-7 cells were transfected with HA-ubiquitin (1 μg), BIM_EL (1 μg) and either 1 or 3 μg (+ or ++, respectively) of pVHL expression plasmid. After 24 h, the cells were lysed and immunoprecipitated with anti-BIM polyclonal antibodies. Polyubiquitinated BIM_EL was detected by immunoblotting the immune complexes with anti-HA monoclonal antibody (the asterisk identifies the IgG heavy chain from the anti-Bim antibody used for the immunoprecipitation). Where indicated, the proteasome inhibitor MG132 (50 μM) was added to the cells 6 h prior to lysis. A portion of each cell lysate taken prior to immunoprecipitation was analyzed by immunoblotting for BIM_EL, pVHL and actin. This experiment has been repeated twice and in each case the presence of pVHL resulted in a decrease in the amount of high molecular weight polyubiquitinated BIM_EL. (C) PRC2 and WT7 cells were cultured in serum-free media overnight and then stimulated with fresh serum-containing media for 0, 5, 15, or 30 minutes. Whole cell lysates were prepared and subjected to immunoblotting with antibodies against BIM and actin. (D) Serum-starved PRC2 and WT7 cells were stimulated with fresh serum-containing media or left in serum-free media for 30 min in the presence or absence of the MEK inhibitor U0126 (50 μM) or phosphatidylinositol 3-kinase inhibitor LY294002 (50 μM). At the end of the treatment period, whole cell lysates were prepared and subjected to immunoblotting with antibodies against BIM and actin. Note the MEK-dependent decrease in BIM_EL levels after serum stimulation in PRC2 cells and the absence of any decrease in similarly treated WT7 cells. Results shown in C and D are representative of those obtained in 3 independent experiments.