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. 2010 Aug 9;190(3):331–345. doi: 10.1083/jcb.201003005

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© 2010 Ong et al.

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Figure 2. — p125A exists as a ternary complex with the Sec13/Sec31A heterotetramer in the cytosol. (A) Characterization of p125A antibodies. Residues 500–758 of p125 were expressed as fusion protein with GST (GST-p125A) and used to raise rabbit antibodies. Affinity-purified antibodies were used for immunoblot analysis. 30 µg of HeLa cell (lanes 1, 3, and 5) and HEK293 cell lysate (lanes 2, 4, and 6) were loaded per lane. The lysates were resolved on SDS-PAGE, transferred to PVDF membrane, and then subjected to immunoblot analysis using anti-p125A antibodies. For the second and third panels, anti-p125A antibodies were preincubated with 500 µg of recombinant GST-Bet3 and GST-p125A, respectively. (B) p125A fractionated with Sec31A and Sec13 in gel filtration. HeLa cell cytosol was subjected to gel filtration in Superose 6 at a flow rate of 0.3 ml/min. Fractions (0.6 ml each) were collected and then TCA precipitated. The proteins were subjected to SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against Sec31A, p125A, Sec23A, and Sec13 as indicated. The arrows indicate the fractions in which the molecular weight markers peaked. (C) Sec31A is co-immunodepleted with p125A. Cytosol was prepared from HEK293 cells transfected to express HA-p125A. The cytosol was subjected to three consecutive cycles of immunodepletion using anti-HA or control anti-GFP antibody-conjugated beads. One tenth of the cytosol was collected and TCA precipitated after each round of immunodepletion. The proteins (along with the cytosol before immunodepletion) were subjected to SDS-PAGE and transferred to PVDF and immunoblotted using antibodies against HA, Sec31A, p125A, Sec23A, and Rab8. Sec31A and total p125A were efficiently and proportionally depleted along with HA-p125A. (D) p125A exists in a multimeric form. myc-p125A and HA-p125A were exogenously expressed in transfected HEK293 cells either singly or in combination as indicated. Cell lysates were subjected to immunoprecipitation with anti-myc (lanes 4–6), anti-HA (lanes 7–9), and control mouse IgG (lanes 10–12). The immunoprecipitates, along with the starting materials, were resolved on SDS-PAGE and analyzed by immunoblot with antibodies against myc, HA, Sec31A, and Sec23A. Molecular size markers are in kD.