- 12-9715-41 - Invitrogen Antibodies AKT1 antibody

Submitted references Combinatorial immunotherapy of N-803 (IL-15 superagonist) and dinutuximab with ex vivo expanded natural killer cells significantly enhances in vitro cytotoxicity against GD2(+) pediatric solid tumors and in vivo survival of xenografted immunodeficient NSG mice.

Chu Y, Nayyar G, Jiang S, Rosenblum JM, Soon-Shiong P, Safrit JT, Lee DA, Cairo MS
Journal for immunotherapy of cancer 2021 Jul;9(7)

Novel cytokine-antibody fusion protein, N-820, to enhance the functions of ex vivo expanded natural killer cells against Burkitt lymphoma.

Chu Y, Nayyar G, Kham Su N, Rosenblum JM, Soon-Shiong P, Lee J, Safrit JT, Barth M, Lee D, Cairo MS
Journal for immunotherapy of cancer 2020 Oct;8(2)

IL-15 negatively regulates curdlan-induced IL-23 production by human monocyte-derived dendritic cells and subsequent Th17 response.

Eken A, Okus Z, Erdem S, Azizoglu ZB, Haliloglu Y, Bicer A, Gur TN, Yilmaz E, Karakukcu M, Altuntas HD, Canatan H
Northern clinics of Istanbul 2019;6(4):379-387

Fc Receptor-Like 1 as a Promising Target for Immunotherapeutic Interventions of B-Cell-Related Disorders.

Yousefi Z, Sharifzadeh S, Yar-Ahmadi V, Andalib A, Eskandari N
Biomarker insights 2019;14:1177271919882351

PRMT5 Modulates Splicing for Genome Integrity and Preserves Proteostasis of Hematopoietic Stem Cells.

Tan DQ, Li Y, Yang C, Li J, Tan SH, Chin DWL, Nakamura-Ishizu A, Yang H, Suda T
Cell reports 2019 Feb 26;26(9):2316-2328.e6

The Bone Marrow Protects and Optimizes Immunological Memory during Dietary Restriction.

Collins N, Han SJ, Enamorado M, Link VM, Huang B, Moseman EA, Kishton RJ, Shannon JP, Dixit D, Schwab SR, Hickman HD, Restifo NP, McGavern DB, Schwartzberg PL, Belkaid Y
Cell 2019 Aug 22;178(5):1088-1101.e15

Crosstalks between mTORC1 and mTORC2 variagate cytokine signaling to control NK maturation and effector function.

Wang F, Meng M, Mo B, Yang Y, Ji Y, Huang P, Lai W, Pan X, You T, Luo H, Guan X, Deng Y, Yuan S, Chu J, Namaka M, Hughes T, Ye L, Yu J, Li X, Deng Y
Nature communications 2018 Nov 19;9(1):4874

MicroRNA-126 deficiency enhanced the activation and function of CD4(+) T cells by elevating IRS-1 pathway.

Chu F, Hu Y, Zhou Y, Guo M, Lu J, Zheng W, Xu H, Zhao J, Xu L
Clinical and experimental immunology 2018 Feb;191(2):166-179

Store-Operated Ca(2+) Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming.

Vaeth M, Maus M, Klein-Hessling S, Freinkman E, Yang J, Eckstein M, Cameron S, Turvey SE, Serfling E, Berberich-Siebelt F, Possemato R, Feske S
Immunity 2017 Oct 17;47(4):664-679.e6

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Normal human peripheral blood cells were unstimulated (orange histogram) or stimulated with Lipopolysaccharide (LPS) Solution (500X) (Product # 00-4976-03) (purple histogram), then intracellularly stained with Anti-Human/Mouse phospho-AKT (S473) PE using the Intracellular Fixation and Permeabilization Buffer Set (Product # 88-8824-00) and protocol. Cell in the lymphocyte (left) and monocyte (right) gates were used for analysis.
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Supportive validation

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Experimental details: NULL
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Supportive validation

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Experimental details: NULL
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Supportive validation

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Experimental details: Fig. 4 Crosstalk between mTORC1 and mTORC2 during NK cell activation. a Immunoblotting to detect the phosphorylation (p-) of S6 at ser235/236 (p-S6 ser235/236 ) and Akt at Ser473 (p-Akt ser473 ) in NKL cells after stimulation with IL-15 (10 ng/ml) (left) or IL-15 (100 ng/ml) (right) for the indicated time periods. beta-Actin was used as an internal control. The data shown are representative of three independent experiments. Uncropped gels are shown in Supplementary Fig. 8 . b Intracellular flow cytometric analysis of p-S6 ser235/236 and p-Akt ser473 in murine splenic NK cells (CD3 - CD19 - NK1.1 + ) after stimulation with IL-15 (100 or 10 ng/ml) for the indicated time periods. The MFI of p-S6 ser235/236 or p-Akt ser473 at each time point was normalized to the baseline reading at the 0-h time point for each individual mouse. Four independent individual mice were used. c - e Flow cytometric analysis and cumulative results for p-Akt Ser473 ( c ), CD122 ( d ), and p-STAT5 Y694 ( e ) in splenic NK cells (CD3 - CD19 - NK1.1 + ) from control versus Raptor NK mice. f Intracellular flow cytometric analysis and cumulative results for p-S6 ser235/236 in total splenic NK cells (CD3 - CD19 - NK1.1 + ) and the indicated subpopulations thereof from control versus Rictor / (top) or control versus Rictor NK (bottom) mice. g Statistical quantification of p-S6 ser235/236 (MFI relative to the untreated control littermates) in splenic NK cells (CD3 - CD19 - NK1.1 + ) wit
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Supportive validation

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Experimental details: Fig. 5 mTORC2 and mTORC1 promote NK cell maturation by controlling the expression of Tbx21 and Eomes in a cooperative and nonredundant manner. a Intracellular flow cytometric analysis and cumulative results for the phosphorylation (p-) of S6 at ser235/236 (p-S6 ser235/236 ) (top) and Akt at Ser473 (p-Akt Ser473 ) (bottom) in splenic NK cells (CD3 - CD19 - NK1.1 + ) from mice of the indicated genotype. b Cumulative ratio and enumeration of NK cells (CD3 - CD19 - NK1.1 + ) in the BM, spleen, and peripheral lymph nodes (pLNs) from mice of the indicated genotype. c Flow cytometric analysis and cumulative frequencies of subpopulations of NK cells (CD3 - CD19 - NK1.1 + NKp46 + ) in the BM, spleen, and pLNs (left) and the calculated ratio of CD27 - versus CD27 + cells among CD11b + NK cells (right). d The cumulative frequencies depicting the CD43 + KLRG1 + subset of NK cells (CD3 - CD19 - NK1.1 + NKp46 + ) in the BM, spleen, and pLNs from mice of the indicated genotype. e , f Tbx21 and Eomes mRNA and protein expression in control versus Rictor / mice ( e ) and control versus Raptor NK mice ( f ), as assessed by quantitative RT-PCR and flow cytometry, respectively. Purified splenic NK cells were used for quantitative RT-PCR (left). Cumulative data for Tbx21 and Eomes protein expression in total splenic NK cells (CD3 - CD19 - NK1.1 + NKp46 + ) and the indicated subpopulations thereof were analyzed by flow cytometry (right). g Cumulative data for Tbx21 and Eomes pr
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Supportive validation

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Experimental details: FIGURE 3 IL-15 exposure downregulates surface Dectin-1 receptor expression and impacts signalling in DCs (A) After five days of culture in with IL-4/GM-CSF or IL-4/GM-CSF/IL-15 DCs were surface stained for Clec7 (Dectin-1) and a representative flow graph and (B) quantification of mean fluorescent intensity (MFI) as bar graph are presented. The experiment was run in triplicate wells and a representative result from a single donor was shown, the experiment was repeated with four different donors. (C) DCs from ""A"" were stimulated with curdlan (50 ug/ml) and phosphorylation of p38, AKT, SRC, NFKB p65, IkBa, ERK1/2 and IRAK4 was measured by phospho-flow assay. A representative result from PBMCs of a single donor was charted. *p
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Supportive validation

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Experimental details: Figure 1 N-803 increased the viability and proliferation of exPBNK with enhanced p-Stat3, p-Stat5, pAKT, p-p38MAPK and NK activating receptors. PBMNCs were stimulated with irradiated genetically modified K562-mbIL21-41BBL cells for 2-3 weeks. (A) Purified exPBNK cells were cultured in complete medium with 0.35 ng/mL (low) or 3.5 ng/mL (high) N-803 or molar equivalent dose of IgG for 3 days. NK viability and proliferation were monitored by MTS assays. The amount of 490 nm absorbance is directly proportional to the number of living exPBNK cells in the culture. The exPBNK cells with N-803 at 0.35 ng/mL or 3.5 ng/mL have significantly higher viability as compared with IgG or medium controls (p
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Supportive validation

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Experimental details: Figure 1 N-820 enhanced the expression of NK activating receptors and proliferation of exPBNK with enhanced Ki67, p-Stat5, and CD25 levels. The percentages of NK activating and inhibitory receptors on exPBNK surface were monitored by flow cytometry analysis at day 3 (A). The NK proliferation was monitored by CellTiter 96 AQueous One solution cell proliferation assay (Promega) according to the manufacturer's instruction (B). Intracellular Ki67 (C), Phosphorylated STAT5 (p-STAT5) (D) and phosphorylated Akt (p-AKT) (E) were monitored by flow cytometry analysis at day 3. Ki67 (p
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. The effect of FCRL1 knockdown the extent of the expression of PI3K /p-AKT pathway in the Ramos and Daudi cells.(a) The real-time PCR approach revealed a significant decrease in theextent of the PI3K gene expression in the BL cellsafter 3 days of the infection procedure. The extent of the expression ofthe p-AKT protein was measured by (b) cytometry and (c) using the FlowJosoftware on day 4 of infection procedure. Shaded-matched representsmatched isotype control antibody, blue represents uninfected cells,green represents control cells, and red represents treated cells. Dataare shown as mean +- standard deviation. BL indicates Burkitt lymphoma; FCRL, Fc receptor-like; PCR, polymerasechain reaction.
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12-9715-41

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