his akt3  (Novus Biologicals)

Journal: bioRxiv

Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

doi: 10.1101/2023.11.21.568047

Figure Lengend Snippet: A ) Schematic shows upstream and downstream kinases in the AKT signaling network. B ) BMDMs were primed, or not, with LPS and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). Phosphorylation of upstream AKT regulators (TBK1, IKKε, PDK1, PTEN and PI3K) was analyzed by immunoblot. Images are representative of three independent experiments. C ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). The phosphorylation of the indicated mTORC1/2-associated proteins was analyzed after 1h by immunoblotting. Data are representative of three independent experiments. D ) WT and Nfe2l2 −/− BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h. The indicated transcripts were analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. Statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparisons test. E-F ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with 15d-PGJ 2 (20, 10 or 1 μM in E and 10 μM in F ) for 1h ( E ) or 24h ( F ). AKT phosphorylation and NRF2 accumulation were assessed by immunoblot ( E ). Data are representative of three independent experiments. IL-10 and TNF release was quantified by ELISA ( F ). n = 6, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Sidak’s test. G ) Expression of Akt isoforms (RNA-seq, Immgen.org database) in the indicated murine macrophage populations. H ) Normalized expression of Akt1 , Akt2 and Akt3 in BMDMs, analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. I, J ) Microscale thermophoresis (MST) analysis of oxPAPC interactions with AKT2 ( I ) and AKT3 ( J ). Traces of fluorescently labeled human recombinant AKT incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes. n = 3, graph shows means ± SD. Images are representative of three independent experiments. K, L ) Active human recombinant AKT2 ( K ) or AKT3 ( L ) were incubated with oxPAPC and a kinase-specific FRET-peptide substrate (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

Article Snippet: His-tagged recombinant human AKT1 (BPS Bioscience, Cat# 40003), AKT2 (BPS Bioscience, Cat# 40011) and AKT3 (BPS Bioscience, Cat# 40012) were fluorescently labeled using His-Tag Labeling Kit RED-Tris-NTA 2 nd Generation (NanoTemper, Cat# MO-L018).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing Assay, Microscale Thermophoresis, Labeling, Recombinant, Incubation, Binding Assay, Derivative Assay, Fluorescence, Inhibition, Blocking Assay