Fig. 8. (A) Co-precipitation of Cnx1p with Sbh1p. A membrane-enriched fraction of the strain expressing the HA-tagged Sbh1p or wildtype was prepared by centrifugation of a total protein extract for 30 minutes at 18,000 g. For solubilization, the pellet was resuspended in phosphate buffer saline and 2% Triton X-100 was added. The sample was further clarified by a centrifugation at 18,000 g. 5 µl of the solubilized supernatant was directly resolved by SDS-PAGE (lane 1) and compared to an anti-HA immunoprecipitate (corresponding to a 100 µl aliquot of the solubilized fraction) from the epitope-tagged strain (lane 2) and from the wild-type strain (lane 3). A western blot analysis was then done using anti-Cnx1p antibodies. (B) Precipitation of Cnx1p with Sec61p or Sec62p. A membrane-enriched fraction of the strain expressing the c-myc-tagged Sec61p was prepared by centrifugation of a total protein extract for 30 minutes at 18,000 g. For solubilization, the pellet was resuspended in phosphate buffer saline and 2% Triton X-100 was added. The sample was further clarified by a centrifugation at 18,000 g. 5 µl of the solubilized supernatant was directly resolved by SDS-PAGE (lane 1) and compared to an anti-c-myc (lane 2) or an anti-Sec62p immunoprecipitate (lane 3) corresponding to a 100 µl aliquot of the solubilized fraction. A western blot analysis was then done using anti-Cnx1p antibodies. (C) Co-precipitation of YlCnx1p with ScSbh2p. A membrane-enriched extract of the strain expressing the ScSbh2p was solubilized in 2% Triton X-100 and centrifuged at 18,000 g for 30 minutes. Proteins in the supernatant were either directly analyzed by SDS-PAGE (lane 1) or first immunoprecipitated in the presence of anti-ScSbh2p antibodies (lane 2) before western blotting using anti-Cnx1p antibodies.

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