human akt1  (BPS Bioscience)

Journal: Frontiers in Pharmacology

Article Title: Integrative analysis of Trichosanthes kirilowii maxim formula granules’ anti-triple-negative breast cancer mechanism via network pharmacology, metabolomics, and molecular pharmacology

doi: 10.3389/fphar.2026.1657396

Figure Lengend Snippet: Molecular docking pattern of schisandrin with target proteins. (A) CCND1; (B) JUN; (C) MYC; (D) AKT1; (E) STAT3; (F) TP53.

Article Snippet: In this study, the recombinant AKT1 protein was obtained from Med Chem Express.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Integrative analysis of Trichosanthes kirilowii maxim formula granules’ anti-triple-negative breast cancer mechanism via network pharmacology, metabolomics, and molecular pharmacology

doi: 10.3389/fphar.2026.1657396

Figure Lengend Snippet: Validation of cell signaling pathways and the AKT1 target. (A,B) Effects of TKM on PI3K/AKT and Wnt/β-catenin signaling pathways and their downstream effectors in MDA-MB-231 cells, as assessed by Western blotting of the indicated signaling proteins, including the pro-apoptotic protein BAX, the anti-apoptotic protein BCL2, and the oncogenic transcription factor c-MYC. (C) Surface plasmon resonance analysis of schisandrin binding to recombinant AKT1 protein. (D) Knockdown efficiency of AKT1 in MDA-MB-231 cells. (E) Effects of schisandrin on the proliferation of control and AKT1-knockdown MDA-MB-231 cells. Data are presented as mean ± SEM of three independent experiments.

Article Snippet: In this study, the recombinant AKT1 protein was obtained from Med Chem Express.

Techniques: Biomarker Discovery, Protein-Protein interactions, Western Blot, SPR Assay, Binding Assay, Recombinant, Knockdown, Control

Journal: Nature Communications

Article Title: Cytoplasmic TRIM24 promotes colorectal cancer cell proliferation by activating Wnt/β-catenin signaling

doi: 10.1038/s41467-025-63685-8

Figure Lengend Snippet: A Kinase prediction tools (KinasePhos 2.0, GPS 3.0, and iGPS 1.0) were used to identify potential kinases targeting TRIM24 at Ser1042. “+” indicates a positive prediction by the respective tool; “−” indicates no prediction. B DLD1 and SW620 cells were transferred with si-NC, si-AURKA, si-AURKB, si-CK2α, and si-AKT. After 72 h, whole cell lysates were derived and immunoblotted for indicated proteins, with Tubulin as a loading control. Experiments were conducted independently three times, consistently producing similar results. C DLD1 and SW620 cells were treated with hesperadin (AURK inhibitor, which has a particularly significant inhibitory effect on AURKB, 20 μM), CX-4945 (CK2 inhibitor, 20 μM), and AKT Inhibitor VIII (AKT1/AKT2 inhibitor, 20 μM) for 4 h and 8 h, respectively. Whole cell lysates were derived and immunoblotted for indicated proteins. Experiments were conducted independently three times, consistently producing similar results. D In vitro phosphorylation assays utilized the purified GST-fusion proteins and AURKB, incubated with hesperadin or DMSO vehicle control. The reaction products were immunoblotted using indicated antibodies. AKT was employed as a positive control. Data are representative of three independent experiments. E SW620 cells were treated with hesperadin for 72 h, whole cell lysates were derived and immunoblotted for indicated proteins. Data are representative of three independent experiments. F Cytosolic (Cyto) and nuclear (Nuc) fractions derived from DLD1 and SW620 cells stimulated with 20 μM of hesperadin for 8 h were immunoblotted for indicated proteins. PARP1 and Caspase-3 served as loading controls and nuclear/cytosolic markers, respectively. Shown are the representative results from three independent experiments. G DLD1-WT/DLD1-KO-1 and SW620-WT/SW620-KO-1 cells were co-transfected with Renilla luciferase and TOPFlash plasmids. The cells were harvested 48 h after transfection, and firefly and Renilla luciferase substrates were added successively. The ratio of the detection value of firefly luciferase to the detection value of Renilla luciferase is taken to compare the difference in the results between the two groups of cells. Data were expressed as means ± SEM, two-tailed Student’s t -test. n = 3 biological replicates per group.

Article Snippet: Recombinant human AURKB, recombinant human AKT1 protein and crystal violet were purchased from Solarbio (China).

Techniques: Derivative Assay, Control, In Vitro, Phospho-proteomics, Purification, Incubation, Positive Control, Transfection, Luciferase, Two Tailed Test

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

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-E ) WT and Tlr4 −/− ( A ), Tlr2 −/− ( B ), Cd14 −/− ( C ), Cd36 −/− ( D ) or Nfe2l2 −/− ( E ) BMDMs were primed, or not, with R848 (1 μg ml −1 ) ( A, C ) or LPS (1 μg ml −1 ) ( B-E ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h ( left, center ) or 24h ( right ). AKT phosphorylation ( A-E ) or NRF2 accumulation ( E ) were assessed by immunoblot ( left ). Data are representative of three independent experiments. ECAR ( center ) was measured using a Seahorse analyzer. n = 6 ( A-C, E ), n = 5 ( D ), data are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. IL-10 and TNF release ( right ) was quantified by ELISA. n = 3, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. F ) oxPAPC binding capacity of AKT was determined by pull down assay. Cellular lysate of 293T cells expressing HA-tagged human AKT1 was incubated with oxPAPE-N-Biotin and the indicated doses of oxPAPC. AKT associated with biotinylated lipids was captured by streptavidin beads and revealed by immunoblotting using anti-HA antibody. β-actin was used as a negative control. Data are representative of three independent experiments. G ) Microscale thermophoresis (MST) analysis of oxPAPC and AKT interactions. Traces of fluorescently labeled human recombinant AKT1 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) ( left and central ). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes ( right ). n = 3, graph shows means ± SD. Images are representative of three independent experiments. H ) Cellular lysate of 293T cells expressing the indicated HA-tagged AKT1 truncated forms were incubated with oxPAPE-N-Biotin. HA-proteins associated with biotinylated lipid were captured by streptavidin beads and revealed by immunoblotting. Data shown are representative of three independent experiments. I ) Active human recombinant AKT1 or PDK1 were incubated with oxPAPC or DPPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, and 1.9 μM) and kinase-specific FRET-peptide substrates (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, Binding Assay, Pull Down Assay, Expressing, Incubation, Negative Control, Microscale Thermophoresis, Labeling, Recombinant, Derivative Assay, Fluorescence, Inhibition, Blocking Assay