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. 2018 Mar 15;29(6):736–750. doi: 10.1091/mbc.E17-08-0518

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© 2018 Liu et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 6: — The cellular distribution of exocyst subunits when Exo70p or Sec3p are irreversibly anchored to the membrane. (A) Total cell lysates (T) and 50,000 × g supernatant (S) and pellet (P) fractions from exo70-sso1TM and tos2TM-sec3 (NY3268 and NY3238) were analyzed by Western blot with anti-Sec10p or anti-Sec15p serum. Heterozygous sec10-sso1TM diploid strain (NY3257, top panel) and sec15-sso1TM diploid strain (NY3258, bottom panel) were used as control. Proteins were detected with anti-Sec10p (top panel) or anti-Sec15p (bottom panel) serum. (B) Yeast strains as indicated (left panel: NY3271, NY3275, NY3276, and NY3277; right panel: NY3276, NY3278, NY3279, and NY3280) were lysed and the 50,000 × g supernatant was used in precipitations with anti-Flag beads. Eluted proteins were detected with anti-Flag, anti-Sec3p, anti-Sec8p, anti-Sec15p, anti-Sec10p, anti-Exo84p, anti-Sec6p, and anti-Exo70p antibodies. Loading was normalized to have an approximately equal amount of the FLAG-tagged subunit, and then the level of all of the other subunits was compared. The relative loading for each sample is listed. Using the same loading ratio, Adhp levels in the 50,000 × g supernatants were examined to reflect the differences in loading.