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. 2016 Jul 21;291(38):20149–20162. doi: 10.1074/jbc.M116.734079

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 7. — OspZ binding to TAB2, TAB3, and ZRANB3 in vitro. A, growth of S. cerevisiae AH109 on medium lacking histidine, adenine, leucine, and tryptophan (QDO) to select for protein-protein interactions (right panel) or medium lacking leucine and tryptophan (DDO) to select for plasmid maintenance only (left panel). Yeast are co-expressing NleE and TAB3 (1), OspZ and TAB3 (2), pGBKT7 and TAB3 (3), NleE and pGADT7 (4), and OspZ and pGBKT7 (5). B, [³H]AdoMet (SAM) labeling of full-length TAB3 immunopurified from HEK293T cells and incubated with purified GST-NleE, GST-NleE^49AAAA52, GST-OspZ, or GST-OspZ^49AAAA52, respectively, in the presence of [³H]AdoMet. [³H]AdoMet labeling was determined by ³H autoradiography (auto). C, FLAG immunoprecipitation (IP) of TAB3-FLAG and detection of EGFP-OspZ and EGFP-OspZ^49AAAA52 in HEK293T cells using anti-GFP antibodies. TAB3-FLAG was detected with anti-FLAG antibodies. Actin is a loading control. D, FLAG immunoprecipitation of TAB2-FLAG and detection of EGFP-OspZ and EGFP-OspZ^49AAAA52 in HEK293T cells using anti-GFP antibodies. TAB3-FLAG was detected with anti-FLAG antibodies. Actin is a loading control. E, FLAG immunoprecipitation of ZRANB3-FLAG and detection of EGFP-OspZ and EGFP-OspZ^49AAAA52 in HEK293T cells using anti-GFP antibodies. TAB3-FLAG was detected with anti-FLAG antibodies. Actin is a loading control.