FIGURE 2.

DT40 cells stably expressing INCENP SAH mutants can proliferate in the absence of endogenous INCENP. A, diagram of INCENP SAH domain swap mutants. Exchange of SAH domains was facilitated by the creation of HindIII, EcoRI, and BamHI sites that did not affect the amino acids sequence. A GFP-TrAP tag was attached to the N terminus of INCENP class I cDNA to visualize the proteins. aa, amino acids. B, expression of INCENP domain swap mutants in stable cell lines 26 h following addition of doxycycline in the medium. Apparently GgINCENP^617–717 is highly antigenic. All our INCENP antibodies have epitopes in this region including the 3D3 antibody (monoclonal antibody against GgINCENP). Consequently, GFP-TrAP-INCENP with the N-terminal half of SAH or MyoM SAH is not detected by the 3D3 monoclonal antibody. The lower panel shows that GFP-TrAP-INCENP with the WT SAH was expressed at levels similar to those of the endogenous INCENP in clone 18 (wild type cells). The upper panel shows that all GFP-TrAP INCENP mutants were expressed at comparable levels except for INCENP with the MyoM SAH, which was expressed at lower levels. In addition, 3D3 antibody recognizes a band at 80 kDa, which is the U35610 PTB-associated splicing factor, which shares some limited peptide sequence with the INCENP coil domain. α-Tubulin (Tub) serves as loading control. C, growth curves of cells expressing INCENP domain swap mutants in the absence of endogenous INCENP. All mutant INCENP cells show no defect in cell proliferation. GFP-TrAP-INCENP^{MyoM SAH}-expressing cells and GFP-TrAP-INCENP^{Double SAH}-expressing cells show a higher percentage of cell death. The average of four independent experiments is shown.