Fig. 4.

Localization of wild-type and CCT-GFP mutants in FOH-treated CHO58 cells. Vectors encoding CCT-GFP or the indicated mutant proteins were expressed in CHO58 cells for 14 h, and shifted to 40°C for 1 h prior to stimulation with 60 μM FOH. After 45 min, cells were fixed and GFP and the nucleus (Hoechst staining) were visualized. A: CCT-GFP and nuclear 2GFP-nuclear localization signal (2GFP-NLS) and cytoplasmic (CCT-Δ29-GFP)-localized controls. B: Domain M point mutants (CCT-8KQ-GFP, -5KQ-GFP, and -3EQ-GFP). C: Phosphorylation mutants (CCT-16SA-GFP, -16SE-GFP, and -7SA-GFP). D: Domain M and P truncation mutants (CCT-ΔM-GFP, -ΔP-GFP, and -ΔMP-GFP). E: Total cell lysates prepared from CHO58 cells, transfected and treated with FOH as described above, were resolved by SDS-PAGE and immunoblotted for GFP and actin.