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. 1998 Mar 9;140(5):1063–1074. doi: 10.1083/jcb.140.5.1063

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Vac8p cosediments with actin filaments. 20 μl (2 mg/ml final concentration) of a high speed, Triton X-100 solubilized total cell extract (A) or cytosol from the myristoylation-minus mutant vac8-2 (B), or cytosol from Δvac8 (C) was mixed with 20 μl fresh G-actin (0.4 mg/ml final concentration) and G-buffer in a 100 μl reaction. G-buffer without actin was added to adjust the volume in the controls. For the experiments using Δvac8 cytosol, 10 μl purified GST-Vac8 protein (26 μg/ml final concentration) was added. The mixtures were incubated for 1 h on ice and then the polymerized actin was pelleted. 5 μl of the supernatant fraction (200 μl final) and the washed pellet fraction (50 μl final) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The same membrane was cut into two halves. The top half was probed with Vac8p antiserum and the bottom half was probed with the antiserum to Yuh1p. In addition, 0.1 μl of the pellet fraction was run separately and used to detect actin.

Vac8p cosediments with actin filaments. 20 μl (2 mg/ml final concentration) of a high speed, Triton X-100 solubilized total cell extract (A) or cytosol from the myristoylation-minus mutant vac8-2 (B), or cytosol from Δvac8 (C) was mixed with 20 μl fresh G-actin (0.4 mg/ml final concentration) and G-buffer in a 100 μl reaction. G-buffer without actin was added to adjust the volume in the controls. For the experiments using Δvac8 cytosol, 10 μl purified GST-Vac8 protein (26 μg/ml final concentration) was added. The mixtures were incubated for 1 h on ice and then the polymerized actin was pelleted. 5 μl of the supernatant fraction (200 μl final) and the washed pellet fraction (50 μl final) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The same membrane was cut into two halves. The top half was probed with Vac8p antiserum and the bottom half was probed with the antiserum to Yuh1p. In addition, 0.1 μl of the pellet fraction was run separately and used to detect actin.