MBC Video and Supplemental Material

This article contains the following material:

• Supplemental Figure
Representative Western blots for quantitation of galactose-induced proteins. A. Amounts of GFP-Net2p from the GAL1 promoter compared to levels of chromosomal Net2p. WT cells containing pKC62, were cultured in Sraf medium and then incubated with galactose for the indicated times to induce expression of GFP-Net2p. Equal amounts of whole cell protein were analyzed by SDS-PAGE followed by Western blotting with the Net2p antibody. Levels of GFP-Net2p (top panel) were normalized to the amount of endogenous Net2p (bottom panel) in each sample. B. Levels of GFP-Net2Np and GFP-Net2WDp at 0'¯ and 180'¯ after induction with galactose. WT cells containing either pKC64 or pKC67 were grown as in A and samples were analyzed using SDS-PAGE followed by Western blotting with the GFP antibody. C. Levels of GFP-Net2WDp and Dnm1p-HA. WT cells containing both pKC68 (pGAL1-GFP-NET2WD) and pHS15 (pGAL1-DNM1) were grown in Sraf medium and then incubated with galactose for 240'¯. Equal amounts of whole cell protein were analyzed by SDS-PAGE followed by Western blotting with either GFP or Dnm1p antibody. Levels of Dnm1p-HA (top panel, arrow) after induction were normalized to levels of endogenous Dnm1p (top panel, arrow head).
• Video.mov (656 KB) 3-D movie of cells expressing both GFP-Net2WDp and Dnm1p from the GAL1 promoter. Localization and distribution of Net2WDp changes when Dnm1p is over-expressed. WT cells expressing both Dnm1p and GFP-Net2WDp from the GAL1 promoter were stained with mitofluor red 589, and examined using the Delta Vision system, capturing 25 sections in the z-plane in 0.2 mum steps. Each section was deconvolved and the resulting images were projected to form a 3-D image that was rotated 180 degrees around the y-axis in 15 degree increments.

Prepared by: Journals Production Manager