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. 2016 Nov 29;45(5):2423–2437. doi: 10.1093/nar/gkw1187

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 1. — Screening and identification the putative proteins interacting with SerRS. (A) The GST pull-down assay. Nuclear proteins extracted from HEK293T were pulled down with the recombinant GST-SerRS(T429A). Lane 1, protein markers; lane 2, protein profiles of GST pull-down; lane 3, protein profiles of GST-SerRS(T429A) pull-down. Protein bands marked with brackets on lane 3 were excised for LC–MS/MS. GST and GST-SerRS(T429A) were marked with ▴ and △, respectively. (B) Screening of three putative proteins interacting with SerRS(T429A) by Co-IP. HEK293T cells were co-transfected with a plasmid expressing Flag-tagged SerRS(T429A) and a plasmid expressing Myc-tagged GATAD2A, HOXB9 or YY1. Cell lysate was immunoprecipitated with anti-Flag (IP: Flag), followed by Western blot (IB) using either anti-Flag to detect Flag-SerRS(T429A) (IB: Flag) or anti-Myc to detect Myc-GATAD2A, Myc-HOXB9 and Myc-YY1 (IB: Myc). A positive band was detected only when co-transfecting Flag-SerRS(T429A) and Myc-YY1 (marked with ▴). Input represents 10% of the total cell extract used for each immunoprecipitation. (C) Co-IP demonstrated the direct interaction between SerRS(T429A) and YY1. HEK293T cells were co-transfected with Myc-YY1 and SerRS(T429A). Afterwards, cell lysate was immunoprecipitated with either anti-Flag (IP: Flag) or anti-Myc (IP: Myc), followed by Western blot using anti-Flag to detect Flag-SerRS(T429A) (IB:Flag) and anti-Myc to detect Myc-YY1 (IB:Myc). Myc-YY1 fusion protein was immunoprecipitated with Flag-SerRS(T429A). Input represents 10% of the total cell extract used for each immunoprecipitation. (D) Quantification of the intensities of Flag and Myc, as shown on Co-IP, when the IP intensities of Flag-SerRS(T429A) and Myc-YY1 were individually normalized as 1. Data are calculated from three independent experiments and presented as mean ± SD (n = 3). Student's t-test was used to determine significant differences between each group (*P < 0.05, **P < 0.01 and ***P < 0.005).