Vps74p is a phospho-protein.
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(A) The anti-Vps74p antibody is specific, Western blot analyses of crude lysates of wild-type (WT) and vps74Δ mutant yeast probed with antibodies against Vps74p (right) and SDS-PAGE stained with Coomassie Blue (left) demonstrates the specificity of the Vps74p antibody. (B) Cell lysis and immunoprecipitation assays were performed as described in the Materials and methods section. Immunoprecipitated proteins were separated into two equal portions and incubated at 37°C in CIP buffer for 1 h in the absence or presence of CIP. Each sample was subjected to SDS-PAGE followed by Western blotting using anti-HA antibody. (C) Schematic representation of the structure of the 346-residue Vps74p polypeptide and various mutant constructs is illustrated. (D) and (E) Expression of wild-type and mutant proteins is dipicted. HA epitope-tagged wild-type (WT) Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm were expressed from 2μ-based plasmids (pVT101U) under the control of the ADH promoter in BY4741 yeast strain and detected by western blotting using the indicated antibodies.