Figure 4.

Characterization of coatomer subcomplexes. (A–C) Coatomer was modified with 1.5 mM or 3.5 mM DMMA, separated by sucrose density centrifugation, and fractionated as shown in Fig. 2. Fractions were dialyzed against 100 mM KTD (pH 6.7) and subjected to immunoprecipitation. (A) Fraction 11 (Fig. 2C) was immunoprecipitated with antibodies against α-COP or β′-COP or with preimmune serum. (B) Fractions 12 and 13 (Fig. 2D) were immunoprecipitated with antibodies against α-COP or ɛ-COP or with preimmune serum. (C) Fractions 13 and 14 (Fig. 2C) were immunoprecipitated with antibodies against δ-COP or ζ-COP or with preimmune serum. The immunoprecipitates of A–C were separated on 7.5% gels under reducing conditions for the detection of β-, α-, β′-, γ-, and δ-COPs and on 15% gels under nonreducing conditions for the detection of ɛ- and ζ-COPs. (D) Coatomer was modified with 1.5 mM DMMA and subjected to Mono Q anion-exchange chromatography as shown in Fig. 5. Fraction 14 was used for immunoprecipitation with anti-β-COP serum or preimmune serum. Immunoprecipitation of the COPs was analyzed by SDS/PAGE on 7.5% gels under reducing conditions Western blotting with antibodies against β-COP and δ-COP.