Tot protein detection and co‐immunoprecipitation

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Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin Tot protein detection and co‐immunoprecipitation. (A) Identification of TOT1–5 as protein‐encoding structural genes. Western analysis: Tot1–5p were tagged individually by one‐step in vivo tagging with a triple c‐Myc tag. Total protein extracts (50 μg) were separated using 10% SDS–PAGE, electroblotted and immunoprobed with the anti‐c‐Myc antibody 9E10. The position of each tagged Tot protein is indicated by an arrow. (B) Tot3p interacts with Tot1p and Tot2p. Protein extracts from strains expressing c‐Myc‐tagged Tot3p together with HA‐tagged Tot1p or HA‐tagged Tot2p were immunoprecipitated using the anti‐c‐Myc antibody 9E10. Precipitates were then separated using 10% SDS–PAGE, electroblotted and immunoprobed with the anti‐HA antibody 12CA5. The positions of HA‐tagged Tot1p and Tot2p are indicated by arrows. (C and D) Tot4p (C) and Tot5p (D) interact with Elp1p (Tot1p), Elp2p (Tot2p) and Elp3p (Tot3p). Protein extracts from strains expressing c‐Myc‐tagged Tot4p (C) or Tot5p (D) were immunoprecipitated using the anti‐c‐Myc antibody 9E10, separated on a 10% SDS–PAGE gel, electroblotted and immunoprobed by western analysis using polyclonal anti‐Elp1p, anti‐Elp2p and anti‐Elp3p antibodies. The positions of co‐immuno precipitated proteins, Elp1p, Elp2p and Elp3p, are indicated by arrows. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 20, Issue: 8, Pages: 1993-2003, First published: 17 April 2001, DOI: (10.1093/emboj/20.8.1993)