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. 2006 Oct;17(10):4364–4378. doi: 10.1091/mbc.E06-02-0143

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© 2006 by The American Society for Cell Biology

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Figure 6. — Golgi membranes from bni1Δ/Δ cells have the same density as wild-type Golgi, but differ in size. (A) Western blot of CaVrg4-HA in hyphal cells. Wild-type or bni1Δ/bni1Δ mutant cells, expressing CaVRG4-HA, were induced to form hyphae, and protein extracts prepared from aliquots collected over time of induction. Equal amounts of protein extract were separated by SDS-PAGE and immunoblotted with anti-HA antibody. (B) Sedimentation equilibrium analysis of Golgi membranes. Protein extracts were prepared from wild-type or bni1Δ/bni1Δ mutant hyphal cells expressing VRG4- HA and subjected to differential centrifugation. The Golgi-enriched P100 fraction was layered on to a 22–60% sucrose gradient, centrifuged at 135,000× g for 18 h. Fractions were removed from the top, subjected to SDS-PAGE, and analyzed by immunoblotting with anti-HA antibodies. (C) Velocity sedimentation analysis of Golgi membranes. The Golgi enriched P100 fraction from wild-type or bni1Δ/bni1Δ cells was layered on to a 5–25% sucrose gradient and centrifuged at 135,000× g for 30 min. Fractions were removed from the top, subjected to SDS-PAGE, and analyzed by immunoblotting with anti-HA antibodies.