Journal: Scientific Reports

Article Title: 3-carbamoyl proxyl nitroxide attenuates CCl 4 -induced liver fibrosis in mice through antioxidant-inflammatory regulation of TLR4/NF-κB signaling pathway

doi: 10.1038/s41598-026-46137-1

Figure Lengend Snippet: Molecular docking analysis of 3-CP with TLR4 and p65. ( A - B ) Predicted 3D binding model of 3-CP and TLR4/p65. 3-CP is colored purple. The protein surfaces of TLR4/p65 are colored in cyan. Potential interactions are indicated by dashed lines. ( C ) Predicted protein-ligand binding affinities (kcal/mol).

Article Snippet: Primary antibodies against NF-κB p65 (Cat #8242S), α-SMA (Cat# 19245 S), collagen I (Cat# 72026 S) and secondary antibody anti-rabbit IgG (CST#7074S) were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Binding Assay, Ligand Binding Assay

Journal: Scientific Reports

Article Title: 3-carbamoyl proxyl nitroxide attenuates CCl 4 -induced liver fibrosis in mice through antioxidant-inflammatory regulation of TLR4/NF-κB signaling pathway

doi: 10.1038/s41598-026-46137-1

Figure Lengend Snippet: 3-CP is associated with inhibition of the TLR4/NF-κB pathway in the CCl 4 -induced liver fibrosis mice model. ( A - B ) Protein expression levels of TLR4/NF-κB signaling-associated proteins (TLR4, MyD88, IKKβ, p65/p-p65 and IκBα/p-IκBα). ( B - C ) Representative results of immunofluorescence staining for p-p65 and p-IκBα (scale bar: 50 μm). ( D ) Quantification of TLR4, MyD88, IKKβ, p65/p-p65 and IκBα/p-IκBα protein expression. Data are presented as mean ± SD, n = 3. * p < 0.05, vs. control group; # p < 0.05, vs. model group.

Article Snippet: Primary antibodies against NF-κB p65 (Cat #8242S), α-SMA (Cat# 19245 S), collagen I (Cat# 72026 S) and secondary antibody anti-rabbit IgG (CST#7074S) were purchased from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Inhibition, Expressing, Immunofluorescence, Staining, Control

Journal: Drug Design, Development and Therapy

Article Title: Cordycepin Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Inhibiting IL-6/IL-6R-Mediated p38 MAPK and NF-κB Activation Through Adenosine A 2A Receptor Stimulation

doi: 10.2147/DDDT.S575035

Figure Lengend Snippet: Cordycepin inhibits IL-6/IL-6R-mediated p38 MAPK and NF-κB phosphorylation activation relying on activation of adenosine receptor A 2A . ( A – E ) Representative blot bands and relative expression levels for IL-6R, NF-κB P-p65, NF-κB p65, NF-κB P-p65/p65, detected by Western blotting. ( F – I ) Representative blot bands and relative expression levels for P-p38 MAPK, p38 MAPK, P-p38/p38 MAPK. ( J – M ) Representative blot bands and relative expression levels for P-STAT3, STAT3, P-STAT3/STAT3. Data were presented as the means ± SEM of three mice in each group and were compared using two-tailed Student’s t tests. # P < 0.05, ## P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. the DSS model (DSS) group.

Article Snippet: Antibodies against NF-κB p65 (8242T) and Phospho-NF-κB p65 (3033T) were obtained from Cell Signaling Technology (CST, USA).

Techniques: Phospho-proteomics, Activation Assay, Expressing, Western Blot, Two Tailed Test, Control

Journal: Drug Design, Development and Therapy

Article Title: Cordycepin Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Inhibiting IL-6/IL-6R-Mediated p38 MAPK and NF-κB Activation Through Adenosine A 2A Receptor Stimulation

doi: 10.2147/DDDT.S575035

Figure Lengend Snippet: The ameliorative effect of cordycepin on DSS-induced colitis is attributed to its activation of A 2A AR, which inhibits IL-6/IL-6R-mediated phosphorylation activation of p38 MAPK and NF-κB.

Article Snippet: Antibodies against NF-κB p65 (8242T) and Phospho-NF-κB p65 (3033T) were obtained from Cell Signaling Technology (CST, USA).

Techniques: Activation Assay, Phospho-proteomics

Journal: Science Advances

Article Title: TLR9-activating cholesterol azetidine derivative–assisted therapeutic vaccines for cancer immunotherapy

doi: 10.1126/sciadv.aeb2465

Figure Lengend Snippet: ( A ) KEGG pathway enrichment analysis of transcriptomic data from BMDCs treated with Aze-Chol NP (20 μg/ml) versus PBS. NOD, nucleotide-binding oligomerization domain. Dot size and color indicate gene count and significance, respectively. ( B ) Heatmap of TLR signaling–related gene expression in BMDCs treated with Aze-Chol NP versus PBS. Z -score normalized values are shown across biological replicates ( n = 3 per group). ( C ) Effect of TLR inhibition on Aze-Chol NP–induced BMDC activation. Flow cytometry analysis of CD80, CD86, and CD40 expression after 1-hour pretreatment with inhibitors against TLR1/2 (CU CPT 22), TLR3 (CU CPT 4a), TLR4 (TAK-242), TLR7/8 (ODN 2088 control), or TLR9 (ODN 2088), followed by Aze-Chol NP stimulation for 24 hours. MFI was min-max normalized. ( D ) Levels of TNF-α, IL-1β, IL-6, and IL-12p70 in supernatants of BMDCs after 1-hour ODN 2088 pretreatment and 24-hour Aze-Chol NP stimulation (20 μg/ml). Data are presented as min-max–normalized values. ( E ) Immunofluorescence validating TLR9 knockout (TLR9-KO) in BMDCs from TLR9-KO versus WT mice. Representative images show TLR9 (green, Alexa Fluor 488), CD11c (red, phycoerythrin), and nuclei (DAPI). ( F ) Flow cytometry analysis of BMDC maturation in WT and TLR9 − / − mice after 24-hour Aze-Chol NP treatment (20 μg/ml). Left: representative flow cytometry plots; right: percentage of mature (CD80 + CD86 + ) BMDCs. ( G ) TLR9-dependent NF-κB activation by Aze-Chol NP assessed using a SEAP reporter assay in HEK-Blue hTLR2, hTLR4, and hTLR9 cells. Optical density at 640 nm (OD 640 ) reflects SEAP activity. ( H ) Molecular docking analysis of the interaction between Aze-Chol and TLR9 protein. ( I ) Activation of MAPK and NF-κB signaling in Aze-Chol NP–treated BMDCs. Representative automated capillary Western blot showing phosphorylated and total levels of NF-κB p65, p38 MAPK, ERK1/2, and SAPK/JNK in BMDCs treated with Aze-Chol NP (20 μg/ml, 4 hours) versus PBS. **** P < 0.0001.

Article Snippet: The primary antibodies (Cell Signaling Technology) were as follows: NF-κB p65 (D14E12) XP rabbit monoclonal antibody (mAb; #8242), phospho–NF-κB p65 (Ser 536 ) (93H1) rabbit mAb (#3033), p44/42 MAPK (ERK1/2) rabbit mAb (#4695), p-p44/42 MAPK(ERK1/2) rabbit mAb (#4370), p38 MAPK (D13E1) XP rabbit mAb (#8690), phospho-p38 MAPK (Thr 180 /Tyr 182 ) (D3F9) XP rabbit mAb (#4511), SAPK/JNK antibody (#9252), phospho-SAPK/JNK (Thr 183 /Tyr 185 ) (81E11) rabbit mAb (#4668), and β-actin (13E5) rabbit mAb #4970.

Techniques: Binding Assay, Gene Expression, Inhibition, Activation Assay, Flow Cytometry, Expressing, Control, Immunofluorescence, Knock-Out, Reporter Assay, Activity Assay, Western Blot