aimp3  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response

doi: 10.1016/j.jbc.2021.101203

Figure Lengend Snippet: MSC association and protein integrity of EPRS-FLAG protein overexpressed in HEK293 T cells. A , doxycycline-inducible EPRS knockdown in HEK293T cells using shEPRS ( left ) or a nonspecific shRNA ( right ). B , immunoprecipitation experiments with FLAG antibody in HEK293T cells with endogenous EPRS knockdown and WT and mutant EPRS-FLAG overexpression. EPRS ( top ) and MSC scaffold proteins AIMP2 ( middle ) and AIMP3 ( bottom ) were visualized by immunoblotting using the indicated antibodies.

Article Snippet: Beads were washed three times with 0.02% PBS-T and boiled in SDS-PAGE loading buffer, followed by immunoblotting with the following antibodies: EPRS (Novus Biologicals NBP1-84929), AIMP2 (Thermo Scientific PA5-31306), AIMP3 (Invitrogen PA5-28283), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bio-Rad).

Techniques: shRNA, Immunoprecipitation, Mutagenesis, Over Expression, Western Blot

Journal: The international journal of biochemistry & cell biology

Article Title: Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 simultaneously binds Glutamyl-Prolyl-tRNA Synthetase and Scaffold Protein Aminoacyl tRNA synthetase complex interacting multifunctional protein 3 of the Multi-tRNA Synthetase Complex

doi: 10.1016/j.biocel.2018.04.015

Figure Lengend Snippet: AIMP1- EGFP a.a. 1-70 fusion protein transiently expressed in HEK293 cell line with myc-EPRS a.a. 1-200 and HA-AIMP2. Validation of transfection efficiency determined by input analysis with WB of EGFP and myc antibodies (A). Co-immunoprecipitation with EGFP and WB analysis of myc and HA (Output). Immuno-precipitation of overexpressed EGFP-N3 vector, AIMP1- EGFP a.a. 1-70 or AIMP1- EGFP a.a. 1-212 was probed for endogenous EPRS and AIMP3 (B). Input and output analysis of GFP confirmed expression. In vitro AIMP1 and AIMP3 interaction used purified recombintant His-tagged AIMP3, GST-control, GST-tagged AIMP1 and GST-tagged AIMP1 a.a. 1-70 purified from bacterial lysates (C). AIMP3 was eluted from nickel beads, while GST fusions left bound to glutathione beads used for pull down. WB analysis was performed using an AIMP3 antibody (D). Surface plasmon resonance using histidine-tagged AIMP1 and AIMP3 purified from bacterial lysates. AIMP1 was immobilized on the chip and AIMP3 was used as a ligand at low concentration (10 fold dilute, 20 μg/mL, Blue line: E) and high concentration (Red line: E, 200 μg/mL) (n=5, representative) with reversible binding demonstrated and an SPR Equilibrium Binding Constant of 25.325 ±01.13×10−13 SD Kd (nM) determined using equilibrium constants where injection of AIMP3 where each point represents a saturation point on the sensogram. Kd values were determined by fitting ≥ 5 points with a nonlinear least squares analysis of the binding isotherm (Req=Rmax/(1+Kd/C). E, is a representative sensogram from the data showing AIMP1/AIMP3 binding responses over time. Equilibrium binding curves of AIMP1/AIMP3 at given concentrations and Kd values determined from an average of 3 separate experiments ± SD (F).

Article Snippet: The samples were resolved on 4–12% gradient gel (Invitrogen) and probed with anti AIMP3 antibodies (Santa Cruz Biotechnology sc-376019) Mass-spectroscopy Mass spectrometry was performed after in-gel tryptic digest using Matrix Assisted Laser Desorption Ionization-time of flight (MALDI-TOF) mass spectroscopy analysis at the University of Notre Dame Mass-spectroscopy and Proteomic facility.

Techniques: Biomarker Discovery, Transfection, Immunoprecipitation, Plasmid Preparation, Expressing, In Vitro, Purification, Control, SPR Assay, Concentration Assay, Binding Assay, Injection