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. 2002 May;22(10):3316–3326. doi: 10.1128/MCB.22.10.3316-3326.2002

FIG. 1.

FIG. 1.

Ubiquitination of Jak2 in response to cytokine stimulation. (A) 32D cells were starved overnight and pretreated with proteasome inhibitor MG132 (20 μM) or vehicle for 1 h before IL-3 (100 ng/ml) stimulation. The cells were lysed in NP-40 lysis buffer supplied with 20 μg of MG132/ml and 20 μg of ubiquitin aldehyde/ml to inhibit isopeptidase activities. Jak2 was immunoprecipitated (IP) and subjected to immunoblotting using an antiubiquitin antibody. After being stripped the filter was reblotted with an anti-Jak2 antibody. (B) 32D cells were treated as described in for panel A. Cell extracts were prepared under denaturing lysis conditions, and the immunoprecipitated Jak2 was analyzed by immunoblotting using an antiubiquitin antibody. (C) Cos-7 cells were starved overnight and pretreated with proteasome inhibitor MG132 (20 μM) or vehicle for 1 h before IFN-γ (100 ng/ml) stimulation. Cell extracts were prepared under denaturing lysis conditions, and the immunoprecipitated Jak2 was analyzed by immunoblotting using an antiubiquitin antibody. After being stripped the filter was reblotted with an anti-Jak2 antibody.