14-4040-82

antibody from Invitrogen Antibodies

Targeting: TRAF3IP2 ACT1, C6orf2, C6orf4, C6orf5, C6orf6, CIKS, DKFZP586G0522

Western blot (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

14-4040-82

Provider

Invitrogen Antibodies

Product name

ACT1 Monoclonal Antibody (9ACT12), eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: The monoclonal antibody 9ACT12 reacts with human Act1 (NF-kappaB activator 1), also known as CIKS (a connection to IkB kinase and stress-activated kinase). Originally cloned for its ability to activate NF-kappaB and its direct interaction with IKKgamma, Act1 has been subsequently shown to play several roles in immunity. In B cells, it is a negative regulator of CD40 and BAFFR signaling affecting reduced survival. This regulation is through direct interaction of Act1 with CD40 or BAFFR and TRAF3. In addition, Act1 is involved in IL-17R signaling. It interacts with IL-17RA through the homotypic SEFIR interaction domain and is critical for downstream NF-kappaB- and C/EBP-mediated inflammatory responses.

Antibody clone number

9ACT12

Concentration

0.5 mg/mL

References

Submitted references

Fusion transcripts FYN-TRAF3IP2 and KHDRBS1-LCK hijack T cell receptor signaling in peripheral T-cell lymphoma, not otherwise specified.

Debackere K, Marcelis L, Demeyer S, Vanden Bempt M, Mentens N, Gielen O, Jacobs K, Broux M, Verhoef G, Michaux L, Graux C, Wlodarska I, Gaulard P, de Leval L, Tousseyn T, Cools J, Dierickx D
Nature communications 2021 Jun 17;12(1):3705

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Natural killer cell functional defects in pediatric patients with severe and recurrent herpesvirus infections.

Ornstein BW, Hill EB, Geurs TL, French AR
The Journal of infectious diseases 2013 Feb 1;207(3):458-68

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IL-17 receptor adaptor protein Act1/CIKS plays an evolutionarily conserved role in antiviral signaling.

Ryzhakov G, Blazek K, Lai CC, Udalova IA
Journal of immunology (Baltimore, Md. : 1950) 2012 Nov 15;189(10):4852-8

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IL-17 receptor signaling inhibits C/EBPbeta by sequential phosphorylation of the regulatory 2 domain.

Shen F, Li N, Gade P, Kalvakolanu DV, Weibley T, Doble B, Woodgett JR, Wood TD, Gaffen SL
Science signaling 2009 Feb 24;2(59):ra8

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The critical role of epithelial-derived Act1 in IL-17- and IL-25-mediated pulmonary inflammation.

Swaidani S, Bulek K, Kang Z, Liu C, Lu Y, Yin W, Aronica M, Li X
Journal of immunology (Baltimore, Md. : 1950) 2009 Feb 1;182(3):1631-40

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The adaptor protein CIKS/Act1 is essential for IL-25-mediated allergic airway inflammation.

Claudio E, Sønder SU, Saret S, Carvalho G, Ramalingam TR, Wynn TA, Chariot A, Garcia-Perganeda A, Leonardi A, Paun A, Chen A, Ren NY, Wang H, Siebenlist U
Journal of immunology (Baltimore, Md. : 1950) 2009 Feb 1;182(3):1617-30

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Deficiency of Act1, a critical modulator of B cell function, leads to development of Sjögren's syndrome.

Qian Y, Giltiay N, Xiao J, Wang Y, Tian J, Han S, Scott M, Carter R, Jorgensen TN, Li X
European journal of immunology 2008 Aug;38(8):2219-28

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoblotting of reduced human peripheral blood mononuclear cell lysates probed with 5 µg/mL of Anti-Human ACT1 Purified. Bands were visualized using Mouse Anti-Rat IgG HRP.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 3 Membrane anchoring of FYN-TRAF3IP2 is required to activate NF-kappaB signaling. a Immunofluorescence images of 293T cells transfected with TRAF3IP2 , FYN-TRAF3IP2 and FYN G2A -TRAF3IP2 stained for TRAF3IP2. Scalebars represent 10 um. b Outgrowth of Ba/F3 cells transduced with pMIG- FYN-TRAF3IP2 or pMIG- FYN G2A -TRAF3IP2 after withdrawal of IL-3. n = 3 biological replicates per condition. c Western blot analysis for TRAF3IP2 on cytoplasmic and membrane fractions of Jurkat cells transduced with pMIG- TRAF3IP2 , pMIG- FYN-TRAF3IP2 and pMIG- FYN G2A -TRAF3IP2 . d NF-kappaB GFP reporter signal intensity in Jurkat cells with empty control vector or expression of TRAF3IP2, FYN-TRAF3IP2 or FYN G2A -TRAF3IP2 after stimulation with an agonistic anti-CD3epsilon antibody or exposure to control immunoglobulin (Ig). n = 3 biological replicates per condition for Ig control, n = 4 for anti-CD3epsilon. p values were calculated with Tukey's post-hoc multiple comparisons test. e Immunofluorescence images (left) of TRAF3IP2 staining on CD4 + T cells transduced to express TRAF3IP2, FYN-TRAF3IP2 or FYN G2A -TRAF3IP2. Scalebars represent 5 um. Quantification (right) of TRAF3IP2 signal intensity in the outer cell bin, expressed as fraction of signal intensity over the entire cell. Each dot represents a cell ( n = 201 for TRAF3IP2, n = 192 for FYN-TRAF3IP2 and n = 179 for FYN G2A -TRAF3IP2). Horizontal line and whiskers represent median and interquartile range respectively. p values were calc

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 4 FYN-TRAF3IP2 activates TRAF6 and canonical NF-kappaB signaling independent of the CBM signalosome. a Western blot analysis of whole-cell lysate and anti-TRAF6 immunoprecipitate in TRAF3IP2-, FYN-TRAF3IP2- and FYN-TRAF3IP2 DeltaT6 -expressing Jurkat cells. b Western blot analysis of whole-cell lysate and anti-TRAF3IP2 immunoprecipitate in TRAF3IP2-, FYN-TRAF3IP2- and FYN-TRAF3IP2 DeltaT6 -expressing Jurkat cells. c Outgrowth of Ba/F3 cells transduced with pMIG- FYN-TRAF3IP2 or pMIG- FYN-TRAF3IP2 DeltaT6 after withdrawal of IL-3. n = 3 biological replicates per condition. d Western blot analysis for canonical NF-kappaB activation in Jurkat cells transduced with pMIG- TRAF3IP2 , pMIG- FYN-TRAF3IP2 or pMIG- FYN-TRAF3IP2 DeltaT6 . e Intracellular flow cytometry for phospho-p65 in resting CD4 + T cells with expression of TRAF3IP2, FYN-TRAF3IP2 or FYN-TRAF3IP2 DeltaT6 . n = 3 biological replicates per condition. p values were calculated with Tukey's post-hoc multiple comparisons test. f NF-kappaB luciferase reporter signal intensity in wild-type Jurkat cells (left) or CARD11 knock-out Jurkat cells (right) with empty control vector or expression of TRAF3IP2, FYN-TRAF3IP2 or FYN-TRAF3IP2 DeltaT6 in resting conditions or after stimulation with PMA. n = 4 biological replicates per condition. p values were calculated with Tukey's post-hoc multiple comparisons test. All data are represented as mean +- SD.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Fig. 5 Expression of FYN-TRAF3IP2 in hematopoietic cells fosters the development of PTCL-NOS. a Kaplan-Meier survival curve after transplantation with HSPC transduced with empty pMIG vector ( n = 5), pMIG- FYN-TRAF3IP2 ( n = 10) or pMIG- FYN G2A -TRAF3IP2-GFP ( n = 6). Log-rank p value was obtained from a two-sided Chi-square test. b Spleen weights at sacrifice of mice transplanted with HSPC transduced with empty vector (EV, n = 5), FYN-TRAF3IP2 (FT, n = 9) or FYN G2A -TRAF3IP2 (F G2A T, n = 6). p values were calculated with Tukey's post-hoc multiple comparisons test. c Representative H&E stains of lymph nodes from mice transplanted with HSPC transduced with empty vector ( n = 5), FYN-TRAF3IP2 ( n = 5) or FYN G2A -TRAF3IP2 ( n = 5). Low magnification images are in the top row, scalebars represent 500 um. High magnification images are in the bottom row, scalebars represent 50 um. d Representative flow cytometry plots for cell suspensions from the lymph nodes of EV mice, FT mice and F G2A T mice (top). Quantification of the lineage of GFP + cells in lymph node cell suspensions (bottom). n = 5 mice per group. p values were calculated with Tukey's post-hoc multiple comparisons test. e Representative H&E stains of livers from mice transplanted with HSPC transduced with empty vector ( n = 5), FYN-TRAF3IP2 ( n = 5) or FYN G2A -TRAF3IP2 ( n = 5). Low magnification images are in the top row, scalebars represent 500 um. High magnification images are in the bottom row, scalebars represe