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. 2002 Mar;22(6):1693–1703. doi: 10.1128/MCB.22.6.1693-1703.2002

FIG. 5.

FIG. 5.

CKI promotes ubiquitination of mPer proteins. (A) To show ubiquitination of mPer proteins, each of Myc-tagged mPer proteins was coexpressed with HA-tagged ubiquitin in the presence or absence of CKIɛ, CKIδ, or CKIɛ(KR). The cell lysates were then subjected to immunoprecipitation with anti-Myc antibody (9E10). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-HA polyclonal antibody. The accumulation of high-molecular-mass species recognized by the anti-HA antibody indicates that Myc-tagged proteins become multiply ubiquitinated. (B) To study the metabolic stability of mPer proteins, each of Myc-tagged mPer proteins was expressed in COS7 cells, together with or without CKIɛ. The cells were incubated in methionine-cysteine-deficient medium and then pulsed with 35S-labeled methionine-cysteine. The cells were incubated for the indicated lengths of time in DMEM-10% fetal calf serum with or without MG-132. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10). The immunoprecipitates were detected with a Bio-Rad phosphorimager.