Fig 1: Two-dimensional MRM profile of ARS proteins in the fractions of size exclusion chromatography. (A) Two cell lysates and affinity purification eluate were injected on a Superdex 200 column and the elution profile was recorded by following the 280 nm absorbance. Red: HEK 293T cell lysate; Blue: KARSoe cell lysate; Green; KARSoe-AP eluate. (B, C and D) The average amount of 25 ARS proteins in 20 fractions of HEK 293T (B), KARSoe (C) and KARSoe-AP (D) from duplicated LC-MRM runs is represented as heat maps. The average value is normalized against the largest value among the 20 fractions (S2, S3 and S4 Tables). In the heat maps, each row represents an ARS protein; each column represents an SEC fraction. (D, right panel) The histogram represents recovery rate (%) of each ARS protein after affinity purification. The recovery rate is a relative value to that of KARS which was used as a bait.
Fig 2: 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.
Fig 3: Overall workflow for detection of ARS proteins. (A) Overall workflow. Two cell lysates (HEK 293T and KARSoe) and affinity purification eluate (KARSoe-AP) were fractionated using SEC. Each fraction was digested with trypsin and spiked with the SIS peptide. After C18 clean-up, the digest was analyzed by LC-MRM-MS. All the data were analyzed by Skyline. (B) Overexpression of KARS tagged with S/FLAG/SBP in HEK 293T cell was detected by western blot using anti-FLAG antibody. Actin was used as a loading control. N, HEK 293T cell; Koe, KARS-overexpressing cell.
Fig 4: TARSL2 as a member of ARS core complex.(A, B) TARSL2 and TARS were detected in AIMP1, AIMP2, and KARS immunoprecipitates of HEK 293T (A) and HCT-8 cells (B). EPRS was used as a positive control. Actin was used for loading control. TCL; total cell lysate, SA pull down; streptavidin pull down. (C) Endogenous EPRS was co-immunoprecipitated with TARSL2. Cell lysate (500 μg) was immunoprecipitated with the antibodies against TARSL2, TARS, and IgG and probed for EPRS, TARSL2 and TARS. (D) Reciprocal co-immunoprecipitation. Cell lysate (500 μg) was immunoprecipitated with the antibodies against EPRS, AIMP1, AIMP2, KARS and IgG and probed for TARSL2. IgG was used for immunoprecipitation control. M; Mock, E; ERPS, A1; AIMP1, A2; AIMP2, K; KARS, In; Input, TL2; TARSL2, T; TARS, IgG(R); rabbit IgG, IgG(M); mouse IgG. Closed arrowheads (▶) indicate TARSL2 and TARS.
Fig 5: Network analysis of AIMP1, AIMP2 and KARS interactome in HEK 293T cells.Network analyses for AIMP1 (A), AIMP2 (B) and KARS (C) interactome were conducted by STRING 9.05. Red dotted box indicates ARS core-complex comprising AIMPs, D, EP, I, K, L, M, Q and RARS. Ribosomal proteins are grouped in grey dotted box. Blue dotted box shows protein group of proteosome and black dotted box showed tubulin proteins. The baits are indicated with black circles with yellow circumference.
Supplier Page from Abcam for Anti-LysRS antibody [EPR7921]