FIG. 7.

Interactions of the complex containing Pob3-Nhp6a and Cdc68. (A) An Nhp6a function of the Pob3-Nhp6a protein depends on Cdc68 association. Cells of the Δnhp6a/b POB3-NHP6A-V5-His₆ strain NB6251 harboring the pRS424 vector (lane 1) or the POB3 plasmid p424-HAPOB (lane 2) or the POB3 + NHP6A plasmid pHAPNV5 (lane 3) were grown at 37°C in enriched selective medium to ∼10⁷ cells/ml. Whole-cell extracts were treated with anti-Cdc68 antibody, and the immunoprecipitates were resolved (8% gel) and analyzed for coimmunoprecipitation of Pob3-Nhp6-V5 and HA-Pob3 using anti-V5 and anti-HA antibodies. On the same blot (but in different lanes), whole-cell extracts (input) were probed with anti-V5 antibody to indicate the relative amounts of extract used for immunoprecipitation. (B) The assemblage of Cdc68 and Pob3-Nhp6a protein binds Nhp6a. Anti-Cdc68 immunoprecipitates from whole-cell extracts of Δnhp6a/b POB3 cells (strain RJY6249) and Δnhp6a/b POB3-NHP6A-V5-His₆ cells (strain NB6251), each housing p314-NV5 (lanes 2 and 4) or the pRS314 vector (lanes 1 and 3), were resolved (6-10-15% gel) and analyzed by Western blotting with anti-V5 antibody.