Fig. 7. The patient-derived Q360* mutation disrupts nuclear localization of SPOP and impairs the SPOP-mediated poly-ubiquitination and degradation of NANOG.

a Dynamic monitoring of the proliferation of SW1990 cells after transfection with indicated plasmids using the iCELLigence RTCA analyzer. **P < 0.01; ***P < 0.001. b Western blot analysis of SW1990 proteins separated into nuclear (N) and cytoplasmic (C) fractions. The SW1990 cells were transfected with the indicated plasmids for 48 h and separated into nuclear and cytoplasmic fractions. Histone H3 and β-actin served as nuclear and cytoplasmic markers, respectively. c Representative fluorescence photomicrographs displayed co-localization (white) of SPOP (green) and NANOG (red) in the nucleus (blue). The SW1990 cells were transfected with the indicated plasmids for 24 h. d Western blot analysis of whole-cell lysates from SW1990 cells transfected with indicated plasmids. e SW1990 cells were transfected with indicated plasmids. Co-IP was performed to study NANOG-SPOP interaction after treatment with 20 μM MG132 for 8 h. f His-NANOG and HA-Ub were expressed in SW1990 cells with wild-type or mutant SPOP. Cells were treated with 20 μM MG132 for 8 h. Ubiquitination assays were performed to study the effect of SPOP mutants on NANOG ubiquitination