Journal: Advanced Science

Article Title: VDLIN: A Deep Learning‐Based Platform for Methylcobalamin‐Inspired Immunomodulatory Compound Screening

doi: 10.1002/advs.202413775

Figure Lengend Snippet: Co7 induces Ifnb1 expression via the TLR4 signaling pathway. A) Volcano plot illustrating the distribution of differentially expressed genes (DEGs) between the Co7 and DMSO treatment groups after 3 h in RAW 264.7 cells. Fold changes are presented as log 2 transformations. Red dots represent DEGs upregulated in the Co7 group. B) Protein‐protein interaction (PPI) network analysis of differentially expressed genes (DEGs) in the Co7‐treated group compared to the DMSO group, revealing significant enrichment in pathways associated with the innate immune response and type I interferon signaling. C) KEGG pathway enrichment analysis of DEGs induced by Co7, highlighting associations with innate immune response pathways. D) Co7 (50 µmol/L) exhibiting strong antiviral effects against VSV in RAW 264.7 and HT29 cells. E) Co7 significantly reduced the inflammatory response induced by LPS, VSV, EMCV, and HSV in RAW 264.7 cells. F) Volcano plot representing the differential gene expression analysis between Co7‐ and LPS‐treated RAW 264.7 cells after 3 h of treatment. Fold changes are displayed as log 2 transformations. Red dots indicate genes upregulated in the Co7 group, while blue dots represent downregulated genes compared to LPS treatment. G) Western blot analysis demonstrating that Co7 inhibited the expression of iNOS and COX2, as well as the phosphorylation of NF‐κB‐P65 at the protein level in RAW 264.7 cells. H) Co7 significantly reduced the mortality rate in mice (n = 10 per group) following LPS challenge (20 mg/kg), compared to the PBS control group. RT‐qPCR data were presented as means ± SEM from three independent experiments. Statistical significance was determined using one‐way ANOVA with Bonferroni's multiple comparisons test (left three panels in E), paired‐samples t‐test (D, right panel of EMCV and HSV in E), or the log‐rank test (H). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Each inhibitor was dissolved in anhydrous DMSO and diluted to its respective working concentration: C29 (10 μM, S6597, Selleck) served as a TLR2 inhibitor; Procyanidin B1 (30 μM, HY‐N0795, MedChemExpress) acted as a TLR4 inhibitor; MyD88‐IN‐1 (30 μM, HY‐149992, MedChemExpress) was used to inhibit MyD88; Pepinh‐TRIF TFA (30 μM, HY‐P2565, MedChemExpress) functioned as a TRIF inhibitor; and GSK8612 (5 μM, T5540, TargetMol) inhibited TBK1/IKKε.

Techniques: Expressing, Gene Expression, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR

Journal: Neuropsychiatric Disease and Treatment

Article Title: Shisandra Decoction Alleviates Parkinson’s Disease Symptoms in a Mouse Model Through PI3K/AKT/mTOR Signalling Pathway

doi: 10.2147/NDT.S476969

Figure Lengend Snippet: AKT and mTOR phosphorylation and relative abundance of p70S6K. ( A ) WB electrophoretogram. ( B ) and ( C ) Relative degree of AKT and mTOR phosphorylation. ( D ) Relative abundance of p70S6K. Use the Tukey HSD test statistical method. *P < 0.05, **P < 0.01 compared to control. # P < 0.05, ## P < 0.01 compared to model group. Δ P < 0.05, ΔΔ P < 0.01 compared to LY294002 group.

Article Snippet: MPTP (Shanghai Yuanye Biotechnology Co., Ltd., item number S31504-500mg); LY294002 (MedChemExpress, item number HY-10108); LC3 antibody, AKT antibody, p-AKT antibody, PTEN antibody, PI3K antibody, mTOR antibody, p-mTOR antibody, p70S6K antibody, p62/SQSTM1 antibody, α-syn antibody (Wuhan Biode Biotechnology Engineering Co., Ltd., item numbers: BM4827, BM1612, BM4762, A00006-1, PB0351, BM4182, BM4840, M01475-4, PB0458, M00215-3, respectively).

Techniques: Phospho-proteomics, Control

Journal: American Journal of Cancer Research

Article Title: NRSN2 promotes osteosarcoma cell proliferation and growth through PI3K/Akt/MTOR and Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: NRSN2 regulates PI3K/Akt/GSK3β axis and Wnt/β-catenin signaling in osteosarcoma cells. A. The level of phosphorylated Akt, mTOR, p-GSK3β and nuclear β-catenin (nu-β-catenin) are positively correlated with the level of NRSN2 in U2OS and MG63 cells. B. Luciferase reporter assays revealed that NRSN2 could regulate Wnt/β-catenin signaling in U2OS and MG63 cells. C. Knockdown NRSN2 inhibits the expression of CCND1 and c-myc in U2OS cells. D. Overexpression of NRSN2 elevates the mRNA levels of CCND1 and c-myc in MG63 cells. E. The pro-proliferation effect was reversed when treated with IWR-1-endo, a inhibitor of β-catenin. *p<0.05, **p<0.01.

Article Snippet: The following antibodies were used in this study: NRSN2 (1:1000, Proteintech), GAPDH (1:5000, Proteintech), p-Akt, Akt, p-mTOR, mTOR, p-GSK3β, GSK3β, β-catenin and the second antibodies were purchased from Cell Signaling Technology and with 1:1000 dilutions.

Techniques: Luciferase, Expressing, Over Expression

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Increases of key necroptosis proteins in steatotic livers. Wild-type mice were fed with chow diet (CD) or Western diet (WD) for 5 to 12 weeks. Liver steatosis, as assessed by staining with hematoxylin and eosin (H&E; A) and oil red O (B). C: Western blot analysis for receptor-interacting serine/threonine-protein kinase (Ripk)-1, Ripk3, and mixed lineage kinase domain-like protein (Mlkl) after total protein extraction from liver homogenates. D: Densitometry analysis. Data are expressed as means ± SEM. n = 3 per group. ∗P < 0.05 and ∗∗P < 0.01 versus CD.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Western Blot, Staining, Protein Extraction

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Hepatic ischemia-reperfusion (IR) injury in Mlkl wild-type (WT) and knockout (KO) mice fed with chow diet (CD) or Western diet (WD). A–C:Mlkl KO and matched WT mice were fed with CD or WD for 8 weeks and were subjected to 45 minutes of ischemia and 6 and 24 hours of reperfusion. Liver injury as assessed by staining with hematoxylin and eosin (A) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; B), with the area of necrosis with TUNEL-positive staining measured (C). D: Serum alanine aminotransferase (Alt) levels. E: Expression of mixed lineage kinase domain-like protein (Mlkl) as detected by Western blot and densitometry analysis after extraction of plasma membrane fractions from livers. F: Monomer, oligomer, and polymer of Mlkl on Western blot and densitometry analysis after total protein extraction from liver homogenates and gel electrophoresis under nonreducing conditions. Data are expressed as means ± SEM. n = 4 to 7 per group. ∗P < 0.05 versus WT.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay, Expressing, Extraction, Clinical Proteomics, Membrane, Polymer, Protein Extraction, Nucleic Acid Electrophoresis

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Detection of neutrophil infiltration in Mlkl wild-type (WT) and knockout (KO) mouse livers after ischemia-reperfusion (IR) surgery. Mlkl KO and matched WT mice were fed with chow diet (CD) or Western diet (WD) and subjected to 45 minutes of ischemia and 6 and 24 hours of reperfusion. A: Neutrophil staining with myeloperoxidase on liver sections after 6 and 24 hours of reperfusion. B: Mpo-positive cells were counted in 20 high-power fields (HPFs). C: mRNA level of a neutrophil marker Ly6G as analyzed by real-time RT-PCR. Data are expressed as means ± SEM. n = 4 to 7 per group. ∗P < 0.05 versus WT.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Western Blot, Staining, Marker, Quantitative RT-PCR

Journal: The American Journal of Pathology

Article Title: Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)–Mixed Lineage Kinase Domain-Like Protein (MLKL)–Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers

doi: 10.1016/j.ajpath.2019.03.010

Figure Lengend Snippet: Detection of inflammation in ischemia-reperfusion (IR) livers of Ripk3 knockout (KO) mice. mRNA levels of cytokines tumor necrosis factor (Tnf)–α (A), Il-1β (B), and Il-6 (C), and chemokine macrophage inflammatory protein (Mip)-2 (D) as analyzed by real-time PCR. Total protein and plasma membrane fractions were extracted from livers. Equal amounts of proteins from the samples of the same groups were combined. E–G: Expression levels of mixed lineage kinase domain-like protein (Mlkl), receptor-interacting serine/threonine-protein kinase (Ripk)-1, and Ripk3 in total lysate (E); plasma membrane Mlkl (F); and oligomer and polymer of Mlkl (G) as detected by Western blot analysis. Data are expressed as means ± SEM. n = 4 to 7 per group. ∗P < 0.05 versus wild type (WT). #Nonspecific band. CD, chow diet; Gapdh, glyceraldehyde phosphate dehydrogenase; WD, Western diet.

Article Snippet: Antibodies used in this study were Mlkl and β-actin (catalog numbers SAB1302339 and A5441, respectively; Sigma-Aldrich, St. Louis, MO); glyceraldehyde phosphate dehydrogenase (Gapdh), Ripk1, and phosphorylated (p)-Mlkl (catalog numbers 2118, 3493, and 62233; Cell Signaling Technology, Beverly, MA); pMlkl, sodium potassium ATPase, and proteasome subunits (Psm)-α2 and -β5 (catalog numbers ab66596, ab76020, ab109525, and ab3330; Abcam, Cambridge, MA); Ripk3 (catalog number 2283; ProSci, Poway, CA); myeloperoxidase (Mpo; catalog number PP023AA; Biocare Medical, Concord, CA); and 26S proteasome regulatory subunit 4 (Psmc1; catalog number A303-821A; Bethyl Laboratories, Montgomery, TX).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Clinical Proteomics, Membrane, Expressing, Polymer, Western Blot

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mTOR Human Recombinant Protein , Origene , Cat# TP320457.

Techniques: Western Blot, Transduction, In Situ, Recombinant, Lysis, Protease Inhibitor, Staining, Magnetic Beads, Clone Assay, Bicinchoninic Acid Protein Assay, Isolation, Labeling, Viability Assay, RNA Sequencing Assay, Sequencing, Expressing, CRISPR, Mass Spectrometry, Mutagenesis, shRNA, Plasmid Preparation, Luciferase, Software, Blocking Assay

Journal: Cell Death & Disease

Article Title: Microglia as modulators of exosomal alpha-synuclein transmission

doi: 10.1038/s41419-019-1404-9

Figure Lengend Snippet: Whole-cell lysates were analyzed by western blot. a Protein levels of LC3 ( n = 5, ** p = 0.0082), P62/SQSTM1 ( n = 5, **** p < 0.0001), Beclin ( n = 5, * p = 0.0101), phospho-AKT ( n = 5, *** p = 0.0004), total-AKT, phospho-mTOR ( n = 4, * p = 0.0183), and β-actin were shown in indicated cells. b Whole-cell lysates were immunoblotted against α-syn (oligomeric) ( n = 5, ** p = 0.0078); monomeric ( n = 4, * p = 0.0113) and β-actin for control. c The triple-stained image of P62/SQSTM1 (gray) with IBA1 (green) and DAPI (blue) in the SNpc

Article Snippet: The following primary antibodies were utilized: rabbit antibody to IBA1 (1:1000, 10904-1-AP, Proteintech), mouse antibody to tsg 101 (1:500, sc-7964, Santa cruz biotechnology), mouse antibody to CD63 (1:1000, ab59479, Abcam), rabbit antibody to Calnexin (1:1000, 10427-2-AP, Proteintech), rabbit antibody to α-syn (1:1000, ab138501, ab52168, Abcam), rabbit antibody to Oligomer (1:1000, AHB0052, ThermoFisher scientific), rabbit antibody to TH (1:1000, 25859-1-AP, Proteintech), rabbit antibody to LC3 (1:1000, 14600-1-AP, Proteintech), rabbit antibody to SQSTM1/P62 (1:1000, ab91526, Abcam), rabbit antibody to Beclin1 (1:1000, 11306-1-AP, Proteintech), rabbit antibody to phosphor-AKT (1:800, AP0655, Abclonal), rabbit antibody to AKT (1:800, A11030, Abclonal), rabbit antibody to phosphor-mTOR (1:400, BM4840, Boster), mouse antibody to α-syn (1:1000, synuclein Ab-2, Thermo Scientific), and rabbit antibody to p-syn (1:500, GTX50222, Genetex).

Techniques: Western Blot, Control, Staining

Journal: bioRxiv

Article Title: Argon inhibits autophagy and improves cerebral ischemia-reperfusion injury via PI3K/Akt/mTOR pathway

doi: 10.1101/2025.10.31.685964

Figure Lengend Snippet: A: western blot results showed that argon treatment significantly upregulated the levels of p-PI3K, p-Akt, and p-mTOR. B: Bar chart for A; C: western blot results of PI3K/Akt/mTOR pathway factors and autophagy factors in N 2, N 2 combined with SC79, and N 2 combined with MHY1485 group; D: Bar chart for C. E: western blot results of in argon, argon combined with MK2206 and argon combined with rapamycin groups. F: Bar chart for E. MHY: MHY1485; Rapa: rapamycin.

Article Snippet: The first antibodies included: P62 (Abcam, 1:1000, rabbit derived polyclonal antibody, ab91526), LC3 (Sigma Aldrich, 1:500, rabbit derived polyclonal antibody, ABC929), β-actin (MCE, 1:5000, mouse monoclonal antibody, HY-P80438), PI3K (Abcam, 1:1000, rabbit derived monoclonal antibody, ab302958), p-PI3K (Abcam, 1:1000, rabbit derived polyclonal antibody, ab138364), Akt (MCE, 1:2000, rabbit monoclonal antibody), p-Akt (MCE, 1:1000, rabbit monoclonal antibody, HY-P80276), mTOR (MCE, 1:1000, rabbit polyclonal antibody, HY-P80276), p-mTOR (MCE, 1:1000, rabbit monoclonal antibody, HY-P80837).

Techniques: Western Blot