aimp3 antibodies  (Santa Cruz Biotechnology)

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

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 1. Loss of AIMP3 is a feature in bladder cancer and AIMP3 promoter methylation is cancer-specific. (a) AIMP3 TMA-IHC. Top: Normal urothelium demonstrating high levels of cytoplasmic AIMP3 expression (L 320; R 3100). Bottom: AIMP3 is not expressed in an invasive T2G3 tumour (MIBC) (L 320; R 3100). In A, the dashed square indicates the area at a higher magnification (3100) in the photomicro- graphs on the right (R). (b) Distribution of AIMP3 expression levels across tumours of different stages and grades and normal tissues. There is a reduction in AIMP3 expression in cancer as compared to normal bladder tissue. A downward-shift in the percentage of moderate/high expressing tumours exists with stage/grade progression. (c) Promoter methylation of AIMP3 was detected in about 10% of bladder tumours (both MIBC and NMIBC) but was undetected in normal tissues. (d) Relative AIMP3 mRNA expression stratified by tumour stage/grade and methylation status. Methylated tumours demonstrated a significant loss in expression, while a marked difference was also observed between Ta/T1 and T2/higher tumours (p < 0.001). Bars, SE. [Color figure can be viewed in the online issue, which is available at wileyon- linelibrary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: Methylation, Expressing

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 2. Association between AIMP3 expression, Tp53 transactivity and genomic stability. (a) Relative TP53TG1 mRNA expression stratified by AIMP3 expression levels and TP53 mutation status. Low expressing AIMP3 wt TP53 tumours presented with a loss of TP53TG1 expres- sion comparable to that of mutant Tp53 tumours. Bars, SE. wt, wild-type. (b) Relative AIMP3 expression stratified by FGA. FGA of all tumours were defined as a quantitative expression of genomic instability, assessed by 1-Mb aCGH. Tumours with high levels of AIMP3 exhibited a lower fraction of genome altered (FGA) (p < 0.001). Bars, SE. (c) Dose–response curves of cell lines HeLa, T24, 253J, RT112 and RT4 irradiation (0–5Gy). Bars, SD. (d) AIMP3 protein expression increased with time upon exposure to IR as assessed by western blots and normalised with b actin. Bars, SD (e) Confocal immunofluorescence imaging showing subcellular localisation of AIMP3 in RT4 cells without irradiation (IR2) and with irradiation. (IR1) (3600) (AIMP3-FITC (green-yellow); Actin-TRITC (red-pink); Nuclear DAPI staining (blue); and merged images). Following IR, there is increased nuclear localisation of AIMP3 (Manders coefficient: 0.362 without irradiation com- pared to 0.778 with irradiation). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: Expressing, Mutagenesis, Irradiation, Western Blot, Imaging, Staining

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 3. Reduction of AIMP3 in vitro is associated with increased radioresistance. (a) Western Blot demonstrating significant siRNA- mediated knockdown of AIMP3 between 24 and 96 hrs post-transfection. (b) AIMP3-knockdown cells demonstrate a significant increase in clonogenic survival following irradiation (p < 0.05*). Bars, SD. Relative to cells exposed to IR at IC50 (vertical-banded bars) and those with just transfection-medium exposure with IR at IC50 (horizontal-banded bars), those with AIMP3-knockdown and exposed to IR at IC50 (grey, unbanded bars) have a higher clonogenic survival. Untreated cells were used as reference (100%) for relative changes in clonogenic sur- vival (bars not shown). (c) Clonogenic assay images demonstrating stained and counted colonies in the respective cell lines. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: In Vitro, Western Blot, Knockdown, Transfection, Irradiation, Clonogenic Assay, Staining

Journal: International journal of cancer

Article Title: Loss of expression of the tumour suppressor gene AIMP3 predicts survival following radiotherapy in muscle-invasive bladder cancer.

doi: 10.1002/ijc.29022

Figure Lengend Snippet: Figure 4. Reduced expression of AIMP3 is associated with reduced survival following radiotherapy and higher risk of recurrence. (a) AIMP3 expression in the BCON TMA. Example of AIMP3-negative (top) and AIMP3-positive (bottom) tumours that were treated with either radical radiotherapy alone or radical radiotherapy supplemented with carbogen and nicotinamide. (b) Kaplan-Meier plot illustrates that the adjusted HR for death in AIMP3-positive group was half the HR for death in the AIMP3-negative group in the BCON cohort. (c) Kaplan Meier plot depicts the HR for death in patients with tumour recurrence following radiotherapy was 8.8 relative to patients without recurrence. (d) AIMP3 expression was evaluated on a radical cystectomy TMA set. Kaplan Meier plot demonstrates no significant differences in the HR for death between the AIMP3-positive and negative groups. [Color figure can be viewed in the online issue, which is available at wileyonlineli- brary.com.]

Article Snippet: The slides were incubated with antibodies against AIMP3 (1:25) (Atlas Antibodies, Sigma-Aldrich, UK) at room temperature for 1 hr.

Techniques: Expressing