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. 1999 Nov;10(11):3643–3659. doi: 10.1091/mbc.10.11.3643

Figure 1.

Figure 1

Physical association of AP subunits in AP-1 and AP-2R complexes. (A) Immunoprecipitation of Apl1p- and Apl2p-containing complexes. Polyclonal antibody was used to precipitate the β subunits, Apl1p (lane 2) and Apl2p (lane 3), from nondenatured lysates of Apl4p-HA-expressing cells (GPY 1359). Also analyzed was a sample of total extract from Apl4p-HA cells corresponding to 1/100 of the extract used for the immunoprecipitations (lane 1). Precipitates and total extract were subjected to SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was cut into strips and immunoblotted with 12CA5 antibody to detect Apl4p-HA (upper panels) and polyclonal antibodies to Aps1p and Aps2p (lower panels). (B) Immunoprecipitation of Apm1p-containing complexes. Monoclonal 12CA5 antibody was used to precipitate Apm1p-HA (lanes 2, 3, 5, and 6) from lysates of wild-type (GPY 1100; lanes 2 and 5) or Apm1p-HA-expressing (GPY 1415; lanes 3 and 6) cells. A sample of total extract from Apl4p-HA cells corresponding to 1/100 of the extract used for the immunoprecipitations was also analyzed (lanes 1 and 4). Samples were treated as described in A. 12CA5 antibody was used to detect Apm1p-HA, and polyclonal antibodies were used to detect Apl1p, Apl2p, Aps1p, and Aps2p. (C) Effect of apl3Δ on immunoprecipitation of AP-2R complexes. Coimmunoprecipitations using polyclonal antibody to precipitate Apl1p (lanes 1 and 3) or Apl2p (lanes 2 and 4) from lysates of wild-type (GPY 1100; lanes 1 and 2) or apl3Δ (GPY 1329; lanes 3 and 4) strains were analyzed as in B. (D) Immunoprecipitation of Apm4p- and Apm1p-containing complexes. 12CA5 antibody was used to precipitate Apm4p-HA (lane 1) and Apm1p-HA (lane 2) from lysates of Apm4p-HA-expressing (GPY 2231; lane 1) or Apm1p-HA-expressing (GPY 1415; lane 2) strains. Samples were analyzed as in B. (E) Coimmunoprecipitation of Apm2p with Apl2p. 12CA5 antibody was used to precipitate Apl1p (lane 1), Apl2p (lane 2), or Apl6p (lane 3) from Apl1p-HA-expressing (GPY 2210), Apl2p-HA-expressing (GPY 2211), or Apl6p-HA-expressing (GPY 2212) strains, all of which carry the multicopy APM2 plasmid. Samples were analyzed as in B. The lower-molecular-weight HA-containing bands in lanes 2 and 3 are degradation products. (F) Immunoprecipitation of Apm1- and Apm2-containing complexes. 12CA5 antibody was used to precipitate Apl2p-HA (lane 1) and Apm1p-HA (lane 2) from Apl2p-HA-expressing (GPY 2211) or Apm1p-HA-expressing (GPY 2213) strains, both of which carry the multicopy APM2 plasmid. Samples were analyzed as in B.