Journal: Microorganisms

Article Title: Monosodium Glutamate Inhibits Pseudomonas aeruginosa -Induced Acute Lung Injury by Targeting the Type III Secretion Systems and Modulating Host Immunity

doi: 10.3390/microorganisms14030725

Figure Lengend Snippet: KEGG pathway enrichment analysis and key protein expression in P. aeruginosa -induced acute lung injury (ALI) in mice. ( A ) KEGG pathway enrichment of differentially expressed genes between the ALI model and control groups; ( B ) protein levels of phospho-JNK, p-p38, p-p65, TLR4, and MyD88 in lung tissues compared between the ALI model and control groups by Western blot analysis (β-actin loading control); ( C ) comparison of protein levels (p-JNK, p-p38, p-p65, TLR4, MyD88) in lung tissues between the model and MSG-treated groups, as detected by Western blot (β-actin control) (n = 12 samples per group; data represent means ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The antibodies for phospho-NF-κB p65 (82335-1-RR), p-65 (80979-1-RR), phospho-P38 (14064-1-AP), phospho-JNK (80024-1-RR), MyD88 (23230-1-AP), TLR-4 (19811-1-AP), P38 (14064-1-AP), JNK (51153-1-AP), and β-actin (66009-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Expressing, Control, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: Validation of the targeting relationship between let-7a-5p and MAPK8. (A) Schematic representation of the predicted complementary binding site between let-7a-5p and the 3′-UTR of MAPK8. (B) Relative mRNA expression level of MAPK8 in macrophages overexpressing let-7a-5p detected by RT-qPCR. (C) WB analysis of MAPK8 protein expression in macrophages overexpressing let-7a-5p. (D) Inhibitory effect of let-7a-5p on MAPK8 expression was assessed via a dual luciferase reporter assay. ( **P < 0.01, ***P < 0.001).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: Biomarker Discovery, Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: Effect of PRP on expression of let-7a-5p and MAPK8. (A) Relative mRNA expression levels of let-7a-5p in knee joint sections of each group detected by RT-qPCR. (B) iNOS and CD206 co-immunolabeld with MAPK8 and counter-stained with DAPI in synovial tissues (Scale bar: 50 μm). (C, D) Quantification of iNOS and CD206 expression co-localized with MAPK8. ( **P < 0.01).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: The let-7a-5p/MAPK8 axis regulates macrophage polarization and inflammatory cytokine release in vitro . (A) IF staining showing expression of iNOS and CD206 in macrophages (Scale bar: 50 μm). (B) Quantification of iNOS-positive cell rate in transfected macrophages. (C) Quantification of CD206-positive cell rate in transfected macrophages. (D-G) Relative mRNA expression levels of pro-inflammatory cytokine (IL-1β and TNF-α) and anti-inflammatory cytokine (IL-4 and IL-10) in transfected macrophages detected by RT-qPCR. ( *P < 0.05, **P < 0.01, ns no significance).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: In Vitro, Staining, Expressing, Transfection, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Constitutively Active Homo-oligomeric Angiotensin II Type 2 Receptor Induces Cell Signaling Independent of Receptor Conformation and Ligand Stimulation

doi: 10.1074/jbc.m500639200

Figure Lengend Snippet: FIG. 1. a, AT2 receptors form homo-oligomerization and up-regulate after serum-free conditions in PC12W cells. Cell membrane was prepared under serum or serum-free conditions, subjected to SDS-gel electrophoresis under non-reducing conditions, and immunoblotted with anti-AT2 receptor antibody. 30 g of protein was used in each lane. Bmax on the AT2 receptors was also analyzed by Scatchard plot analysis. b, AT2-WT-EGFP receptor localized in the cell membrane after 24 h under serum-free conditions. EGFP- and AT2-WT-EGFP receptor-expressing CHO cell lines were grown under serum or serum-free conditions and stained with DAPI to visualize nuclear morphology as assessed by a laser scanning confocal microscope. Arrows indicate AT2-WT-EGFP receptor translocation in the cell membrane. c, AT2-WT-EGFP receptor induced apoptosis after 48 h under serum-free conditions. AT2-WT-EGFP receptor- and AT2-N127G-EGFP receptor-expressing CHO cell lines were grown under serum conditions or for up to 48 h under serum-free conditions, stained with DAPI, and imaged by a digital fluorescent microscope. d, pharmacological intervention in apoptosis mediated by AT2-WT-EGFP receptor and AT2-N127G-EGFP receptor was analyzed by treating cells under serum conditions and with or without 10 M p38 MAPK inhibitor (SB203580) and 1 M caspase-3 inhibitor (DEVD-cmk) for 48 h under serum-free conditions. Data are shown as the percentage of apoptotic cells in three independent experiments as assessed by TUNEL as described under “Experimental Procedures.” Histograms show that AT2-WT-EGFP receptor transfected CHO cells induced apoptosis under serum (A) or after 48 h serum-free conditions (B). 65% of AT2-WT-EGFP receptor transfected CHO cells showed apoptosis after 48 h of serum-free conditions. *, p 0.05 versus serum conditions. t, p 0.05 versus serum-free conditions without treatment. Data are given as mean S.E. (n 4). e, the AT2 receptor formed a homo-oligomer in the cell membrane. Cell membranes were prepared in serum-free conditions for 24 h, subjected to SDS-gel electro- phoresis after being pretreated with () or without ( ) DTT under non-reducing conditions, and immunoblotted with anti-EGFP antibody. 20 mg of protein was used in each lane. Arrows indicate the monomer and dimer species of the receptor. f, AT2-WT-EGFP receptor formed only homo-oligomer, whereas AT2-N127G-EGFP receptor formed both homo-oligomer and monomer in the cell membrane. Cell membranes were prepared in serum () or serum-free ( ) conditions for up to 48 h, subjected to SDS-gel electrophoresis under non-reducing conditions, and immunoblotted with anti-EGFP antibody. 40 g of protein was used in each lane. Arrows indicate the monomer and dimer species of the receptor. Representative immunoblots (a, d, and f) and pictures (b and c) are shown. Three independent determinations were performed, and similar results were observed (a–d and f).

Article Snippet: Materials—The following antibodies and reagents were generously provided as indicated or purchased: AT2 receptor-selective non-peptide antagonist PD123319 (Research Biochemical International) and the p38 MAP kinase inhibitor SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)1H-imidazole (Upstate Biotechnology); the caspase-3 inhibitor Ac-DEVD-chloro methyl ketone (Calbiochem); antiAT2 receptor antibody catalog number sc-7420 (Santa Cruz Biotechnology).

Techniques: Membrane, SDS-Gel, Electrophoresis, Expressing, Staining, Microscopy, Translocation Assay, TUNEL Assay, Transfection, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Silencing Rac1 inhibits the activation of P38 MAPK signal in vitro . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) were detected by Western blot analysis. GAPDH served as the internal reference. All experiments were repeated 3 times and the results are presented as mean ±SD. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vitro, Western Blot, Negative Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Rac1 silencing inhibits the activation of P38 MAPK signal in vivo . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) in each group were detected by Western blot analysis with GAPDH as the internal reference. All experiments were repeated 3 times. The results are presented as mean ±SD. N=6. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vivo, Western Blot, Negative Control