Figure 8. Immunoisolation and protein... | Rockefeller University Press

Figure 8.

$Figure 8. Immunoisolation and protein composition of preperoxisomal vesicles. Immunoisolation of preperoxisomal vesicles was performed as described in Materials and methods. Pex2-3HA and Pex17-3HA were used as anchors for isolation of vesicles from 20S. After antibodies coupled to Sepharose beads were used to capture the ppVs, beads were washed three times, and associated membranes were eluted in SDS sample buffer. The immunoisolate was analyzed with specified antibodies. The experiment was performed with cells harvested after 6 h in methanol medium. (A) Immunoisolation was performed with 20S fractions from WT, pex1Δ, and pex6Δ cells expressing either Pex17-3HA and Pex2-GFP or Pex2-3HA and Pex17-GFP expressed from the constitutive GAP promoter. In pex3Δ cells, Pex2-GFP was not detectable in the 20S fraction, probably because it is retained in the ER (Fig. 1 C) and thus was not included in the experiment. pex1Δ cells expressing PAOX-Pex1 were switched from YPD to methanol medium (+Methanol) for 6 h or were continued in YPD (−Methanol) before immunoisolation of vesicles. (B) Immunoisolation was performed with 20S fractions from WT, pex1Δ, and pex6Δ cells expressing either Pex17-3HA or Pex2-3HA. The expression of Pex2-3HA was considerably lower than that of Pex17-3HA in pex1Δ, pex6Δ, and pex3Δ cells, and the blot (right) was exposed for a longer duration (∼10×). The experiment was repeated more than three times with similar results.$

Immunoisolation and protein composition of preperoxisomal vesicles. Immunoisolation of preperoxisomal vesicles was performed as described in Materials and methods. Pex2-3HA and Pex17-3HA were used as anchors for isolation of vesicles from 20S. After antibodies coupled to Sepharose beads were used to capture the ppVs, beads were washed three times, and associated membranes were eluted in SDS sample buffer. The immunoisolate was analyzed with specified antibodies. The experiment was performed with cells harvested after 6 h in methanol medium. (A) Immunoisolation was performed with 20S fractions from WT, pex1Δ, and pex6Δ cells expressing either Pex17-3HA and Pex2-GFP or Pex2-3HA and Pex17-GFP expressed from the constitutive GAP promoter. In pex3Δ cells, Pex2-GFP was not detectable in the 20S fraction, probably because it is retained in the ER (Fig. 1 C) and thus was not included in the experiment. pex1Δ cells expressing P_AOX-Pex1 were switched from YPD to methanol medium (+Methanol) for 6 h or were continued in YPD (−Methanol) before immunoisolation of vesicles. (B) Immunoisolation was performed with 20S fractions from WT, pex1Δ, and pex6Δ cells expressing either Pex17-3HA or Pex2-3HA. The expression of Pex2-3HA was considerably lower than that of Pex17-3HA in pex1Δ, pex6Δ, and pex3Δ cells, and the blot (right) was exposed for a longer duration (∼10×). The experiment was repeated more than three times with similar results.