Figure 4.

Nem1-Spo7 complex formation of spo7Δ cells expressing Spo7 with LLI mutations. A, the spo7Δ mutant (GHY68), which harbors chromosomal NEM1-PtA, was transformed with pGH448 and its derivatives for the expression of the WT and mutant forms of Spo7-Myc. The yeast transformants were grown at 30 °C in SC-Leu medium to the late logarithmic phase, and cell extracts were prepared. The cell extracts were adjusted to a protein concentration of 2.5 mg/ml and incubated with IgG-Sepharose. The affinity resins were precipitated, washed, and treated with the Laemmli sample buffer. After centrifugation, the affinity-purified proteins in the supernatant were resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was cut at the 50-kDa position, and the upper portion was probed with anti-protein A antibody, whereas the lower portion was probed with anti-Spo7 antibody. The positions of Nem1-PtA, Spo7-Myc, and molecular mass standards are indicated. B, the signals of Nem1-PtA and Spo7-Myc in panel A were quantified by ImageQuant software. The levels of mutant Spo7-Myc were normalized to the level (set at 100%) of the WT protein; the levels of Nem1-PtA from the transformant cells expressing mutant Spo7-Myc were normalized to the protein level (set at 100%) from those expressing the WT protein. The density of a background region on the blot was subtracted from the density of the protein band of interest. The immunoblots in panel A are representative of three separate experiments, whereas the data in panel B are averages from the three experiments ± S.D. (error bars). The individual data points are also shown. *, p < 0.05 versus Nem1-PtA of cells expressing WT Spo7-Myc. #, p < 0.05 versus Spo7-Myc of cells expressing WT Spo7-Myc.