Figure 4.

Spc94p/Nud1p, Cnm67p, and Spc42p are present in common complexes. (A) A lysate of cells expressing SPC94–ProA (lane 1) was separated by centrifuging (40,000 × g, 30 min, 4°C) into a cytoplasmic extract (lane 2) and a SPB-containing pellet (lane 3). This pellet was extracted with 1 M NaCl/1% Triton X-100/50 mM Tris⋅HCl (pH 7.5) followed by centrifuging, resulting in a supernatant (lane 4) and a pellet (lane 5). Both extracts (lanes 2 and 4) were incubated with IgG Sepharose. The bound proteins were eluted (lanes 7 and 9). Lanes 6 and 8 are controls of wild-type cells that were treated identically to lanes 7 and 9, respectively. Fractions were tested by immunoblotting using antibodies directed against the indicated proteins. (B) Two-hybrid interactions between Spc42p, Cnm67p, and Spc94p/Nud1p. Experiment was performed as described in Fig. 2B legend.