Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex
Figure 1
Affinity purification of SBP-tagged AIMP1, AIMP2 and KARS.
(A) Schematic diagram of AIMP1, AIMP2, and KARS constructs for affinity purification. S/FLAG/SBP tags were attached to the N-terminus of cloned genes. (B) Expression of AIMP1, AIMP2, and KARS tagged with S/FLAG/SBP in HEK 293T cells were confirmed by immunoblotting analysis using anti-FLAG antibody. Closed arrowheads (◀) indicate AIMP1, AIMP2 and KARS. (C) Streptavidin affinity purification was carried out and 10 % of the eluted samples from HEK 293T and HCT-8 cells were visualized by protein staining. One of three biological replicates is shown and the bait proteins are marked with red arrows. (D) 90 % of elution was separated on SDS-PAGE to about 1-cm distance and divided into three fractions each. Then, tryptic peptides were recovered from each gel bands and analyzed by LC-MS/MS. SAINT algorithm was used to calculate the likelihood of true interaction of identified proteins. M; Mock, A1; AIMP1, A2; AIMP2, K; KARS. ‘Mock’ is a vector having the S/FLAG/SBP tag only without target genes.
