14-7888-82

antibody from Invitrogen Antibodies

Targeting: IRF8 ICSBP, ICSBP1, IRF-8

Western blot (Supportive data in Antibodypedia)

Flow cytometry (Recommended by provider)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

14-7888-82

Provider

Invitrogen Antibodies

Product name

IRF8 Monoclonal Antibody (GW4CML3), eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: The GW4CML3 monoclonal antibody reacts with human and mouse interferon regulatory factor 8 (IRF8; ICSBP). IRF8 is a 50 kDa transcription factor that plays a critical role in the development of dendritic cells (DC). Specifically, IRF8 is essential in the development and differentiation of plasmacytoid DC (pDC) and CD8a+ DC. IRF8-/- mice are deficient in both pDC and CD8a+ DC populations. CD8a- DC are present in normal numbers in IRF8-/- mice but fail to mature upon Toll-like receptor signaling and are functionally impaired in response to in vivo microbial stimulation. IRF8 deficiency does not affect the frequency or viability of DC precursors and as a result retroviral IRF8 transduction restores pDC development from DC progenitors in IRF8-/- mice. Applications Reported: This GW4CML3 antibody has been reported for use in intracellular staining followed by flow cytometric analysis, and western blotting. (Fluorochrome conjugated GW4CML3 is recommended for use in intracellular flow cytometry). Applications Tested: This GW4CML3 antibody has been tested by western blot on lysates from stimulated normal human peripheral blood cells. This can be used at 1-5 µg/mL. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.

Reactivity

Human, Mouse

Host

Mouse

Isotype

IgG

Antibody clone number

GW4CML3

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

4° C

References

Submitted references

Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion.

Gangoso E, Southgate B, Bradley L, Rus S, Galvez-Cancino F, McGivern N, Güç E, Kapourani CA, Byron A, Ferguson KM, Alfazema N, Morrison G, Grant V, Blin C, Sou I, Marques-Torrejon MA, Conde L, Parrinello S, Herrero J, Beck S, Brandner S, Brennan PM, Bertone P, Pollard JW, Quezada SA, Sproul D, Frame MC, Serrels A, Pollard SM
Cell 2021 Apr 29;184(9):2454-2470.e26

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IRF8 mutations and human dendritic-cell immunodeficiency.

Hambleton S, Salem S, Bustamante J, Bigley V, Boisson-Dupuis S, Azevedo J, Fortin A, Haniffa M, Ceron-Gutierrez L, Bacon CM, Menon G, Trouillet C, McDonald D, Carey P, Ginhoux F, Alsina L, Zumwalt TJ, Kong XF, Kumararatne D, Butler K, Hubeau M, Feinberg J, Al-Muhsen S, Cant A, Abel L, Chaussabel D, Doffinger R, Talesnik E, Grumach A, Duarte A, Abarca K, Moraes-Vasconcelos D, Burk D, Berghuis A, Geissmann F, Collin M, Casanova JL, Gros P
The New England journal of medicine 2011 Jul 14;365(2):127-38

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Novel markers of normal and neoplastic human plasmacytoid dendritic cells.

Marafioti T, Paterson JC, Ballabio E, Reichard KK, Tedoldi S, Hollowood K, Dictor M, Hansmann ML, Pileri SA, Dyer MJ, Sozzani S, Dikic I, Shaw AS, Petrella T, Stein H, Isaacson PG, Facchetti F, Mason DY
Blood 2008 Apr 1;111(7):3778-92

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Essential role for ICSBP in the in vivo development of murine CD8alpha + dendritic cells.

Aliberti J, Schulz O, Pennington DJ, Tsujimura H, Reis e Sousa C, Ozato K, Sher A
Blood 2003 Jan 1;101(1):305-10

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Cutting edge: IFN consensus sequence binding protein/IFN regulatory factor 8 drives the development of type I IFN-producing plasmacytoid dendritic cells.

Tsujimura H, Tamura T, Ozato K
Journal of immunology (Baltimore, Md. : 1950) 2003 Feb 1;170(3):1131-5

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Cell lysates prepared from normal peripheral blood cells were treated for 16 hours with LPS and IFN gamma (left lane) or left untreated (right lane) and were immunoblotted with 5 µg/mL of Anti-Human/Mouse IRF8 Purified antibody.Bands were visualized using Anti-Mouse IgG HRP.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Cell lysates prepared from normal peripheral blood cells were treated for 16 hours with LPS and IFN gamma (left lane) or left untreated (right lane) and were immunoblotted with 5 µg/mL of Anti-Human/Mouse IRF8 Purified antibody.Bands were visualized using Anti-Mouse IgG HRP.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Cell lysates prepared from normal peripheral blood cells were treated for 16 hours with LPS and IFN gamma (left lane) or left untreated (right lane) and were immunoblotted with 5 µg/mL of Anti-Human/Mouse IRF8 Purified antibody.Bands were visualized using Anti-Mouse IgG HRP.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-IRF8 Monoclonal Antibody (GW4CML3), eBioscience™(Product # 14-7888-82) and a 50kDa band corresponding to IRF8 was observed across cell lines and tissue extract tested except K-562, T98G and Mouse Skeletal Muscle which are reported to be negative. Nuclear enriched extracts (30 µg lysate) of THP-1 (Lane 1), Raji (Lane 2), Ramos (Lane 3), K-562 (Lane 4), T98G (Lane 5) and tissue extracts of Mouse Skeletal Muscle (Lane 6) and Mouse Spleen (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2.5 ug/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure S5 Irf8 is responsive to IFNgamma and TAMs in NPE and PPP cells in vitro and is important for immune evasion, related to Figure 6 (A) Immunoblot analysis of Irf8 expression in NPE cells and subsequently tumor-derived lines with and without in vitro IFNgamma treatment (representative of n = 3 experiments). (B) RT-qPCR analysis of Irf8 ( left ) and Ifih1 ( right , confirms stimulation of IFN signaling) expression in untreated (UT) NPE, NPE-BL6-TD and NPE-IE cells versus in vitro treatment with IFNalpha/beta (a, b, respectively). Error bars represent SEM, RQ (relative quantification) relative to NPE untreated conditions. (C) Schematic of Irf8-mCherry reporter design. (D) Flow cytometric analysis of mCherry expression in NPE-Irf8-mCherry reporter lines +- IFNgamma treatment versus Parental NPE lines with adjunct histograms showing population distributions. (E) Gating strategy employed to isolate GFP + tumor cells, CD45 + CD3 + T cells and CD45 hi/lo F4/80 + myeloid cells from NPE tumors in NSG or BL6 host mice. Related to Figure 6 E. (F) RT-qPCR analysis of selected target gene expression ( H2-Ab1 , H2-Q7 and Ifi47 ) in 'immune naive' NPE cells in vitro compared with cells derived directly from NPE tumors in NSG and BL6 hosts. Each point represents technical duplicates of cells isolated from individual animals. Related to Figure 6 E (G) Heatmap illustrating expression levels of selected target gene expression ( Irf8 , H2- Q10, H2 -Ab1 , H2-Q7 and Ifi47 ) in untreated NPE c

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 6 Irf8 is responsive to IFNgamma and TAMs in NPE cells in vitro and is important for immune evasion (A) GSEA of IFNgamma response signature in NPE-IE cells (enrichment score [ES] and FDR reported). (B) RT-qPCR analysis of Irf8 expression in NPE, NPE-BL6-TD and NPE-IE cells in vitro +- IFNgamma treatment. (C) Representative immunoblot of IRF8 expression in NPE cell panel +- IFNgamma time series treatment in vitro (n = 3). (D) Representative immunoblot of IRF8 and pSTAT1 expression in NPE cell panel with IFNgamma/Tofacitinib treatment in vitro (n = 3). (E) qRT-PCR analysis of Irf8 expression in NPE cells in vitro versus GFP + cells derived directly from NPE tumors in NSG/BL6 hosts. Points represent technical duplicates of cells isolated from individual animals. (F) Schematic of NPE cells co-culture with immune populations derived from NPE tumors. (G) RT-qPCR analysis of Irf8 expression in NPE cells co-cultured as in (F) (paired t test). (H) Representative bioluminescent imaging of NPE-IE/NPE-IE- Irf8 KO tumor progression in BL6 in vivo ( Irf8 KO clone G4 shown). (I) Survival of BL6 mice orthotopically transplanted with NPE-IE cells versus clonally derived NPE-IE- Irf8 KO lines (NPE-IE, n = 12; NPE-IE Irf8 KO G4, n = 15; NPE-IE Irf8 KO K6, n = 10; NPE-IE Irf8 KO E6, n = 6). p values for survival of each Irf8 KO line versus parental NPE-IE: ** parental versus Irf8 KO G4, p = 0.0047; * parental versus Irf8 KO K6, p = 0.0323; parental versus Irf8 KO E6, p = 0.0857. RT-qPCR d