MA1-513

antibody from Invitrogen Antibodies

Targeting: AHR bHLHe76

Provider product page for MA1-513

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Gel shift

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [24]
Comments [0]
Validations
Immunocytochemistry [3]
Flow cytometry [2]
Other assay [12]

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Product number: MA1-513 - Provider product page
Provider: Invitrogen Antibodies
Product name: AHR Monoclonal Antibody (RPT9)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA1-513 detects the aryl hydrocarbon receptor (AhR) from human, mouse, and rat cells. MA1-513 has been successfully used in immunoprecipitation, chromatin immunoprecipitation, immunocytochemistry, immunohistochemistry, immunofluorescence, and gel shift procedures. This antibody specifically immunoprecipitates a single ~95 kDa protein representing AhR from Hepa 1 cytosol. Although MA1-513 can be used in Western blotting procedures, MA1-514 (clone RPT1) is recommended for this procedure. The MA1-513 immunogen is a synthetic peptide corresponding to residues R(12) K R R K P(17) V(22) K P I P A E G I K(31) of mouse AhR.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: RPT9
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

AHR protein structure - MA1-513 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Aryl Hydrocarbon Receptor using Aryl Hydrocarbon Receptor Monoclonal Antibody (RPT9) (Product # MA1-513) shows staining in A2058 Cells. Aryl Hydrocarbon Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Aryl Hydrocarbon Receptor (Product # MA1-513) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Aryl Hydrocarbon Receptor using Aryl Hydrocarbon Receptor Monoclonal Antibody (RPT9) (Product # MA1-513) shows staining in Hela Cells. Aryl Hydrocarbon Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Aryl Hydrocarbon Receptor (Product # MA1-513) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Aryl Hydrocarbon Receptor using Aryl Hydrocarbon Receptor Monoclonal Antibody (RPT9) (Product # MA1-513) shows staining in U251 Cells. Aryl Hydrocarbon Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Aryl Hydrocarbon Receptor (Product # MA1-513) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Total amounts of endogenous AhR in the cytosol and nucleus of HeLa cells. (A) HeLa cells were treated with 0.05% DMSO (-) or 1 muM beta-NF (+) for 0, 15, 30, 60, and 120 min at 37 degC. The lysate was collected and centrifuged for 60 min at 105,000 g , then the supernatant was collected. AhR in the cytosol were analyzed by immnoblotting with an anti-AhR antibody or an anti- beta-actin antibody ( n = 4, * p < 0.05). The AhR/beta-actin ratio was quantified using Image J software (Drop of Wisdom). (B) HeLa cells were treated with 0.05% DMSO (-) or 3 muM beta-NF (+) for 30 min at 37 degC. The lysate was collected and centrifuged, then the supernatant was collected. Immunoprecipitation was performed using an anti-Ahr antibody or anti-HSP90 antibody followed by immunoblotting with an anti-HSP90 antibody or an anti-AhR antibody. In panel, the HeLa cell lysate (left two lanes) or immunoprecipitated samples (right two lanes) were separated by SDS-PAGE followed by immnoblotting. The subunit molecular weight of HSP90 and AhR are 90 and 110 kDa, respectively. The 50- and 32-kDa protein bands were the IgG heavy- and light chain, respectively. M, molecular marker proteins. C, HeLa cells were treated with 0.05% DMSO (-) or 1 muM beta-NF (+) for 0, 15, 30, 60, 120, and 360 min at 37 degC. The lysate was collected and centrifuged for 60 min at 105,000 g , then the supernatant was collected as a cytosol fraction. Or the lysate in 2.2-M sucrose solution was centrifuged for 60 min at 30,0

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 beta-NF induced AhR and HSP90 nuclear translocation in HeLa cells. HeLa cells were treated with beta-NF (5 muM) or DMSO (0.1%) for 120 min. Complex formation between HSP90 and AhR was measured by in situ PLA using rabbit anti-AhR antibodies, mouse anti-HSP90 antibodies, and corresponding reagents. In situ PLA is indicated by red signals. Blue staining indicates DAPI staining of cell nuclei. Control was stained with non-immune rabbit- and mouse serum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Supportive validation

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Experimental details: Figure 4 AhR transcriptionally activates miR-142a to inhibit IRF1 and HIF-1alpha expression. (A) The top 30 miRNAs in BMDMs that are regulated by LPS are arranged in a miRNA array heatmap. In addition, miRNAs predicted to be under the transcriptional control of AhR (according to analysis with the JASPAR database) are noted. (B) Venn diagram analyses were performed to identify miRNAs that can both target IRF1 and HIF-1alpha and that are under the transcriptional control of AhR. (C) Renal expression of mmu-miR-142a-3p in mice (n = 6) with CaOx nephrocalcinosis following treatment with an AhR neutralizing antibody or FICZ treatment was assessed via FISH (200x; scale bar: 20 um). (D) qRT-PCR was performed to measure mmu-miR-142a-3p expression in BMDMs using U6 RNA as a normalization control. (E, F) ChIP assays and ChIP qPCR analysis showed that AhR bound to the miR-142a promoter in BMDMs treated with the AhR overexpression plasmid. (G) A schematic model showed that AhR directly binds to the miR-142a promoter and activates its transcription. (H, I) WT and mutated miR-142a targeting sequences in the IRF1 and HIF-1alpha 3'-UTR regions that were used to construct luciferase reporters, with reporters bearing these IRF1 (J) or HIF-1alpha (L) 3'-UTR sequences co-transfected along with miR-142a mimic (100 nM). IRF1 (K) and HIF-1alpha (M) mRNA levels were detected via qRT-PCR in BMDMs following miR-142a mimic or inhibitor transfection. Western blotting (N, O) analysis enabled the detectio

Supportive validation

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Experimental details: Figure 2 ( a ) Western blot analysis of AHR protein expression in HEK293FT (1) and HeLa (2). A band represents the AHR was observed at 95kD as expected. ( b ) Vector map of pGlow-CYP1A1 prom ::mScarlet plasmid.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Anakinra colocalizes with AhR in Il1r1 -/- cells. ( A ) Immunofluorescence imaging of cellular localization of FITC-anakinra in alveolar macrophages, isolated from naive C57BL/6 or Il1r1 -/- mice, primed with 100 ng/mL LPS for 2 hours at 37degC, and exposed to 10 mug/mL FITC-anakinra for 60 minutes. ( B ) Immunofluorescence analysis of FITC-anakinra in Il1r1 -/- cells exposed to FITC-anakinra at 30 and 60 minutes in the presence of 5 muM cytochalasin D. Insets, percentage positive cells. ( C ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra for 0, 2, and 4 hours and assessed for AhR and IL1Ra expression by immunoblotting with specific antibodies. ( D ) Immunofluorescence imaging of alveolar macrophages from naive C57BL/6 and Il1r1 -/- mice primed with 100 ng/mL LPS for 2 hours at 37degC, exposed to 10 mug/mL FITC-anakinra for 60 minutes, and stained with anti-AhR antibody. ( E ) Immunofluorescence imaging of MEF cells stained with anti-AhR antibody and DAPI and exposed to 10 mug/mL FITC-anakinra for 60 minutes. ( F ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra, lysed, immunoprecipitated with anti-AhR antibody, and assessed for IL1Ra and AhR expression by immunoblotting with specific antibodies. None, untreated cells. Representative images, acquired with a fluorescent microscope (BX51), and immunoblots from 2 independent experiments are shown. DAPI was used to detect nuclei. Sections were examined using a Zeiss Axio Observer Z1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Anakinra activates a xenobiotic sensing pathway via IDO1. ( A ) H1L1 cells were treated with different doses of kynurenine and anakinra for 6 hours and assessed for luciferase assay. ( B ) Representative immunoblots of IL1RA, HSP90, AIP, and AhR and ( C ) ARNT and AhR in cell lysates in which AhR was immunoprecipitated from Il1r1 -/- MEF cells treated with 10 mug/mL anakinra or 10 muM FICZ. In B and C , data are representative of 1 out of 2 independent experiments and the relative densitometric analysis are reported. ( D and E ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra for 2 and 8 hours and analyzed for gene expression by a custom QuantiGene plex gene expression assay. Fold changes are reported as heatmap for 2 and 8 hours ( D ) and histograms for 8 hours ( E ) (data are representative of 1 out of 2 independent experiments). ( F ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra and analyzed for mRNA expression of selected genes by RT-PCR ( n = 3 independent samples) and protein expression of IDO1 and SOCS3 by immunoblotting (representative experiment). The relative densitometric analysis is reported. ( G ) Il1r1 -/- MEF cells were treated with either 10 mug/mL anakinra for different times or 10 ng/mL IFN-gamma as positive control for 48 hours and assessed for kynurenine production by ELISA. ( H ) Il1r1 -/- MEF cells were treated with 10 mug/mL anakinra in the presence or absence of 10 muM epacadostat and assessed for kynurenine production by

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. The cytoplasmic and nuclear localization of AhR in I3C-treated macrophages. The cytoplasmic and nuclear expression of AhR is demonstrated. The nuclear accumulation of AhR was observed upon I3C treatment among MDMs of SLE patients and normal subjects by immunofluorescence staining (A) . Secondary antibody as the isotype control had no detectable fluorescent emission. Blue DAPI-stained nucleus of MDMs were merged with green-coloured AhR-expressing cells (40X magnification). The percentages of AhR accumulation in each combination are quantified (B ). One-way ANOVA with Tukey's post hoc test were used to compare the means of multiple samples. Data of each bar demonstrates means +- SE. p < .05 were considered as statistically significant. * p < .05, ** p < .01, *** p < .001, and **** p < .0001. NS, not statistically significant; Mock, non-treated monocyte-derived macrophages; JAB, Jurkat apoptotic bodies; I3C, indole-3-carbinol.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 UCHL3 maintained the stability of AhR protein through deubiquitination, thereby promoting PD-L1 expression. A , Positive expression of AhR protein in tumor tissues and adjacent normal tissues from NSCLC patients (n = 45) detected by Immunohistochemistry. B , Silencing efficiency of sh-UCHL3 in A549 cells and overexpression efficiency of LV-UCHL3 in PC9 cells detected by qRT-PCR. C , Protein levels of UCHL3 and AhR in A549 cells treated with sh-UCHL3 or in PC9 treated with LV-UCHL3 detected by Western blot analysis. D , Western blot analysis of AhR protein degradation in A549 and PC9 cells following CHX (10 mug/mL) treatment (the left penal) and corresponding statistical analysis of AhR protein stability (the right penal). E , Co-IP detection of exogenous UCHL3 and AhR proteins in HEK293T cells. F-G, AhR protein degradation rate in A549 cells in response to sh-UCHL3 ( F ) or in PC9 cells in response to LV-UCHL3 ( G ) detected by CHX pulse-chase analysis. H , Detection of AhR protein ubiquitination in A549 cells overexpressing UCHL3 or in PC9 cells silencing UCHL3. I , Expression of PD-L1 in tumor tissues and adjacent normal tissues from NSCLC patients (n = 45) detected by qRT-PCR. J , Expression of PD-L1 in A549 cells treated with sh-UCHL3 or in PC9 cells treated with LV-UCHL3 detected by qRT-PCR. K , Silencing efficiency of sh-AhR in A549 cells and overexpression efficiency of LV-AhR in PC9 cells detected by qRT-PCR (the left two panels) and Western blot analysis (the

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: AhR PAS domain binds to HSP90. (A) Domain structure of human AhR. bHLH, PAS, and TAD indicate basic helix-loop-helix, PER-ARNT-SIM, and transcriptional activation binding domain, respectively. In the present study, we constructed and purified AhR-PAS including PAS-A and PAS-B (115-387). (B) Purified GST-PAS domain was analyzed by SDS-PAGE (9% gel). (C) Digested GST-PAS by factor Xa were separated by SDS-PAGE (9% gel). (D) The factor Xa digested AhR-PAS domains (input) were added to Mock resin (Mock) or beta-naphthoflavone (beta-NF) affinity resin. The bound proteins were separated by SDS-PAGE (9% gel) (D) or by immunoblotting using an anti-AhR antibody (E). In panels B and D, asterisk, double asterisks, and triple asterisks indicate uncut GST-PAS domain, cut PAS domain, and cut GST, respectively.