FIG. 4.

A heterologous NLS overrides the CKIɛ-dependent cytoplasmic localization of mPER1. (A and B) HEK 293 cells were transiently cotransfected with constructs encoding either 4HA-CKIɛ or empty vector and Myc-mPER1 (A) or NLS-mPER1 (B). Forty-eight hours posttransfection, the epitope-tagged proteins were visualized with Alexa 488 (green)-conjugated anti-Myc MAb 9E10 and Alexa 594 (red)-conjugated anti-HA MAb 12CA5, and the nuclei were visualized with Hoechst staining. WT, wild type. (C) NLS-mPER1 is still a substrate for CKIɛ. In vitro-translated [³⁵S]methionine NLS-mPER1 was incubated without or with added CKI. The addition of the amino-terminal NLS did not interfere with the kinase-dependent electrophoretic mobility shift.