rabbit polyclonal antibody for lat1  (Novus Biologicals)

Journal: Journal for immunotherapy of cancer

Article Title: Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes.

doi: 10.1136/jitc-2023-006728

Figure Lengend Snippet: Figure 4 AHR activation by kynurenine and its derivatives is enhanced in the absence of tryptophan. (A)HEK293-E cells were treated for 72 hours with kynurenine (80 µM), AHR inhibitor (CH223191, 10 µM) or both in tryptophan-free medium supplemented or not with 80 µM tryptophan in the presence of 10% FBS. CYP1A1 expression was measured by RT-qPCR analysis. (B)HEK293-E cells were treated for 24 hours with different concentrations of kynurenine in tryptophan-free medium supplemented or not with 80 µM tryptophan in medium without FBS. CYP1A1 expression was measured by RT-qPCR analysis. (C)Flow cytometric evaluation of kynurenine uptake in HEK293-E cells. Kynurenine fluorescence in HEK293-E cells treated with 80 µM kynurenine, in the presence or absence of the System L inhibitor, BCH (10 mM) in PBS at 37°C or 4°C. (D.)Kynurenine fluorescence in HEK293-E cells treated with 80 µM kynurenine and different concentrations of tryptophan in PBS for 4 min. (E, F)RT-qPCR analysis (E)and western blot analysis (F) of SLC7A5 expression in wildtype or GCN2-KO HEK293-E cells cultured for 24 hours in tryptophan medium or tryptophan-free medium. The quantification of LAT1 protein expression is shown in the lower panel, each point representing a technical replicate. (G)Scramble or GCN2-KO HEK293-E cells were cultured overnight in tryptophan-free medium supplemented or not with 80 µM tryptophan without FBS. Cells were then collected and incubated with or without 80 µM kynurenine during 4 min in PBS without tryptophan. Kynurenine uptake was measured by flow cytometry analysis of kynurenine fluorescence. (H)RT-qPCR analysis of CYP1A1 expression in wild-type (upper panel) or GCN2-KO (lower panel) HEK293-E cells treated for 24 hours with kynurenine (80 µM) or FICZ (1 µM) in tryptophan-free medium supplemented or not with 80 µM tryptophan in the absence of FBS. (I)HEK293-E cells were treated for 72 hours with 80 µM kynurenine metabolites in tryptophan-free medium supplemented or not with 80 µM tryptophan in the presence of 10% FBS. CYP1A1 expression was measured by RT-qPCR analysis. (J)HEK293-E cells were treated for 24 hours with different concentrations of kynurenic acid in medium with or without tryptophan and without FBS. CYP1A1 expression was measured by RT-qPCR analysis. (K) RT- qPCR analysis of AADAT expression in wildtype or ATF4-KO HEK293-E cells cultured for 72 hours in tryptophan-free medium supplemented or not with 80 µM tryptophan in the presence of 10% FBS. The mRNA levels of different genes were measured by quantitative RT-qPCR and normalized to GAPDH. Mean±SD of technical triplicates from one representative experiment out of three independent experiments, except for panel F and K, which are Mean±SD of technical replicates from one representative experiment out of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. AHR, aryl hydrocarbon receptor; FBS, fetal bovine serum; ns, not significant; PBS, phosphate-buffered saline.

Article Snippet: AHR (clone D5S6H) rabbit monoclonal antibody, ATF4 (clone D4B8) rabbit monoclonal antibody, GCN2 (#3302S) rabbit polyclonal antibody, LAT1 (#5347S) rabbit polyclonal antibody was purchased from Cell Signaling and diluted at 1/1000.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Fluorescence, Western Blot, Cell Culture, Incubation, Flow Cytometry, Saline

Journal: Journal for immunotherapy of cancer

Article Title: Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes.

doi: 10.1136/jitc-2023-006728

Figure Lengend Snippet: Figure 5 AHR upregulation favors in vitro differentiation of CD4+ FoxP3+ regulatory T lymphocytes. (A) Spearman correlation in bulk TCGA RNAseq data between expression of AHR and IDO1 (left column) and TDO2 (right column) per TCGA cancer type. (B) Spearman correlation between expression of the AHR gene and various QUANTISEQ immune-deconvoluted populations per cancer type in the TCGA patient cohort. (C) Allelic summary of the AHR gene in human and mouse lines. (D) Naïve CD4+ CD62L+ T cells were isolated from WT (wild-type) and Ahr-KO mice from C57BL/6 or DBA/2 background, and were cultured for 5 days with anti-CD3 anti-CD28 beads, TGF-β (5 ng/mL) and IL-2 (100 U/mL). The percentage of Treg differentiation was determined by flow cytometry analysis of cell surface CD4 and intracellular Foxp3 expression. (E) RT-qPCR analysis of Ahr expression in Tregs differentiated from CD4+ T cells isolated from wild-type DBA/2 mice in medium containing or not 80 µM tryptophan. The mRNA levels of different genes were measured by quantitative RT-qPCR and normalized to Gapdh. Mean±SD of triplicates from one out of three experiments. (F) Treg differentiation (same as D) of naïve CD4+ CD62L+ T cells isolated from WT or Ahr-KO DBA/2 mice in medium containing or not 80 µM tryptophan. Mean±SD of technical triplicates from one representative experiment out of three independent experiments. (G) Kynurenine fluorescence in differentiated murine Treg cells treated with 80 µM kynurenine in PBS supplemented or not with tryptophan for 5 min. To demonstrate the LAT1-dependent kynurenine cell entry, we pretreated the cells with BCH (10 mM) in some conditions, as indicated. Cells treated without kynurenine were used as a negative control. Mean±SD of technical triplicates from one representative experiment out of two independent experiments. (H) Treg differentiation (same as D) of naïve CD4+ CD62L+ T cells isolated from WT or Ahr-KO DBA/2 mice in medium containing or not 80 µM tryptophan in the presence or absence of 80 µM kynurenine. Mean±SD of technical triplicates from one representative experiment out of three independent experiments. *p<0.05, **p<0.01, ****p<0.0001. AHR, aryl hydrocarbon receptor; IDO1, indoleamine 2,3-dioxygenase 1; ns, not significant; TCGA, The Cancer Genome Atlas; PBS, Phosphate-buffered saline.

Article Snippet: AHR (clone D5S6H) rabbit monoclonal antibody, ATF4 (clone D4B8) rabbit monoclonal antibody, GCN2 (#3302S) rabbit polyclonal antibody, LAT1 (#5347S) rabbit polyclonal antibody was purchased from Cell Signaling and diluted at 1/1000.

Techniques: In Vitro, Expressing, Isolation, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Fluorescence, Negative Control, Saline

Journal: EJNMMI Research

Article Title: In vitro evaluation of ( S )-2-amino-3-[3-(2- 18 F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid ( 18 F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

doi: 10.1186/s13550-023-00988-1

Figure Lengend Snippet: Properties of tumor-related amino acid transporters

Article Snippet: Rabbit anti-human LAT1 polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ATB 0,+ polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc.) was diluted at 1:50, and rat anti-human xCT monoclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan) was diluted at 1:100.

Techniques:

Journal: EJNMMI Research

Article Title: In vitro evaluation of ( S )-2-amino-3-[3-(2- 18 F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid ( 18 F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

doi: 10.1186/s13550-023-00988-1

Figure Lengend Snippet: Overexpression of human amino acid transporters. Western blot analyses were performed using anti-human A LAT1, B ATB 0,+ , C ASCT2, and D xCT antibodies on membrane protein extracts from the positive-control cells, negative-control cells (CHO-K1 cells), control vector-transfected cells (Mock), expression vector-transfected cells, and gene-specific siRNA-transfected cells (upper panel). The positive-control cells were A MCF-7, B T3M-4, C NCI-H460, and D A549 cells. The same blots were also probed with anti-CD98 antibody (middle panel), and anti-Na + /K + -ATPase antibody as a loading control (lower panel).

Article Snippet: Rabbit anti-human LAT1 polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ATB 0,+ polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc.) was diluted at 1:50, and rat anti-human xCT monoclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan) was diluted at 1:100.

Techniques: Over Expression, Western Blot, Positive Control, Negative Control, Plasmid Preparation, Transfection, Expressing

Journal: EJNMMI Research

Article Title: In vitro evaluation of ( S )-2-amino-3-[3-(2- 18 F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid ( 18 F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

doi: 10.1186/s13550-023-00988-1

Figure Lengend Snippet: Immunofluorescent analysis of human amino acid transporters. Immunofluorescent analyses were performed using anti-human A LAT1, B ATB 0,+ , C ASCT2, and D xCT antibodies (green) on the positive-control cells, negative-control cells (CHO-K1 cells), control vector-transfected cells (Mock), expression vector-transfected cells, and gene-specific siRNA-transfected cells. The positive-control cells were A MCF-7, B T3M-4, C NCI-H460, and D A549 cells. Nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining (blue)

Article Snippet: Rabbit anti-human LAT1 polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ATB 0,+ polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc.) was diluted at 1:50, and rat anti-human xCT monoclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan) was diluted at 1:100.

Techniques: Positive Control, Negative Control, Plasmid Preparation, Transfection, Expressing, Staining

Journal: PLoS ONE

Article Title: Striatal Astrocytes Act as a Reservoir for L-DOPA

doi: 10.1371/journal.pone.0106362

Figure Lengend Snippet: Detection of mRNA expression of DAT (A), protein expression of DAT (B), LAT1 (C), 4F2hc (D), and AADC (E) in primary cultured neurons (N) and astrocytes (G) from mesencephalon (MC) and striatum (Str) of rat embryos using RT-PCR assay (A) and western blot analysis (B–E).

Article Snippet: The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% non-fat milk powder at room temperature for 1 h. Then the blots were incubated with goat polyclonal anti-DAT antibody (1∶200, Santa Cruz Biotechnology, Santa Cruz, CA, #K-20), rabbit polyclonal anti-L-type amino acid transporter LAT1 antibody (dilution; 1∶100, Serotec, Oxford, UK, #AHP735), mouse monoclonal anti-4F2hc antibody (dilution; 1∶200, BD Transduction Laboratories, #611516) or rabbit polyclonal anti-aromatic L-amino acid decarboxylase (AADC) antibody (dilution; 1∶500, Protos Biotech Corporation, New York, NY, #CA201 bDCrab) at room temperature for 1 h. After washing with TBS-T (2×5 min), the blots were reacted with donkey anti-goat IgG (Millipore), donkey anti-rabbit IgG (Amersham Biosciences) or donkey anti-mouse IgG (Millipore) secondary antibody conjugated with horseradish peroxidase (dilution; 1∶2,000 or 5,000) at RT for 1 h. Specific signals of proteins were visualized by chemiluminescence using the ECL Western blotting detection system (Amersham Biosciences).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot