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. 2001 May;12(5):1215–1226. doi: 10.1091/mbc.12.5.1215

Figure 2.

Figure 2

Gyp1p is peripherally associated with membranes. (A) Antibody detection of Gyp1p. Equal amounts of total cell extracts prepared from GYP1 pep4 (NY2295) and gyp1Δ pep4 (NY2296) strains were resolved by SDS-PAGE, blotted, and probed with affinity-purified polyclonal antibody raised against recombinant Gyp1p. The positions of size markers (kDa) are indicated. (B) Lysate of NY2295 was loaded at the bottom of a tube containing 35% iodixanol, and centrifuged in a TLA 120.2 rotor at 120,000 rpm for 3 h. Seven fractions were collected from the top. The percentage of total protein in each fraction (▪) and iodixanol concentration of each fraction (●) was determined. The amount of Gyp1p, ADH, and Sncp in each fraction was determined by Western blot. (C) Lysate of NY2295 was mixed with lysis buffer and lysis buffer containing Triton X-100, urea, and sodium carbonate so that the final concentrations were 2% Triton X-100, 4 M urea, and 0.1 M sodium carbonate (pH 11), respectively. The mixtures were incubated on ice for 40 min and then centrifuged at 100,000 × g for 30 min. Equal amounts of pellet and supernatant fractions were analyzed by SDS-PAGE and Western blot. Urea treatment shifted the distribution of Gyp1p toward the supernatant but did not affect the distribution of the integral membrane protein Ssop.