Figure 2.

Ste14p is an integral membrane protein. A crude yeast cell extract from SM3495 (2μ STE14) was prepared and treated with either buffer G, 0.6 M NaCl, 0.1 M Na₂CO₃ (pH 11), 2.5 M urea, or 0.5% Triton X-100. After incubation on ice for 20 min, one-half of each sample was reserved as total lysate (T). The remaining volume was separated into supernatant (S) and pellet (P) fractions by centrifugation at 200,000 × g. After being resuspended to equivalent volumes with Laemmli sample buffer, equivalent amounts of T, S, and P fractions were subjected to SDS-PAGE and transferred to nitrocellulose. The immunoblots were probed with (A) anti-Ste14p antiserum that had been depleted previously of nonspecific antibodies, (B) anti-Pma1p antiserum, (C) anti-Sec23p antiserum, and (D) anti-hexokinase antiserum.