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Fig. 4. Monitoring the effects of phosphorylation on the Sec63p-Sec62p interaction in vivo. (A) Extracts of yeast cells expressing Sec63-ProA or Sec63T654A-ProA were precipitated with IgG-sepharose and the supernatants (Sn) and the precipitates (P) were probed with anti-Sec62p serum. The precipitate was additionally probed with rabbit anti-goat/goat anti-rabbit antibodies. The asterisk (^*) indicates cross-reacting bands of the anti-Sec62p serum. (B) Split-ubiquitin assay. Yeast cells coexpressing Sec63-C_ub-RUra3p or its mutants together with N_ug-Sec62p or N_ug-Ubc6p were grown to an OD₆₀₀ of 1. Four millilitres of these cultures and tenfold dilutions thereof were spotted on plates lacking uracil, tryptophan and histidine to select for the presence of the plasmids. To check for cell numbers dilutions were also spotted on SD+ura plates (not shown).

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