Journal: bioRxiv

Article Title: MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

doi: 10.1101/2023.01.23.524851

Figure Lengend Snippet: a,b) Quantification of nuclear pBrd4 levels by confocal IF with anti-pBrd4 (green) and DAPI staining of primary microglial cells pre-treated with 10 μM C13 (or DMSO) for 1 h and stimulated or not with 1 μg/mL LPS for 4 h. The data and images shown correspond to one experiment (N=20 analyzed images per condition) and are representative of two independent experiments. Scale bar: 10 μm. Student’s t-test (between two groups as indicated), unpaired, one-tailed. c,d) Quantification (c) and representative image (d) of Western blot analyses with anti-pBrd4 and anti-Brd4 (normalized to Brd4 or α-tubulin signal, respectively) in lysates of WT and MOK-KO SIM-A9 cells stimulated with 1 μg/mL LPS for 1 h. Data are from three independent experiments (N=3). Student’s t-test, unpaired, one-tailed. e,f) Quantification (e) and representative images (f) of confocal IF analyses of nuclear pBrd4 levels with anti-pBrd4 (green) and DAPI staining of WT and MOK-KO SIM-A9 cells stimulated or not with 1 μg/mL LPS for 1 h. Signal was enhanced by 50% in all four images in the first row for better visualization. Images are from one out of three independent experiments (N=3). Scale bar: 10 μm. Student’s t-test, unpaired, two-tailed. g) ChIP-PCR assessment of Brd4-binding to specific cytokine promoters. Represented data are relative values (percentage of input) of PCR products for IL-6, IFNβ and TNFα promoters after ChIP assay with either anti-Brd4 or IgG control antibodies with chromatin isolated from WT or MOK-KO cells treated or not with 1 μg/mL of LPS for 1 hour (N=4). Student’s t-test, paired, one-tailed. Data are the mean -/+ S.E.M. from ‘N’ independent experiments, and *P<0.05, **P<0.01 and ***P<0.001. NS: not significant.

Article Snippet: For intracellular labelling of bi-phosphorylated MOK protein (bpMOK), cells were fixed and permeabilized with the Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences, cat. 554722) according to the manufacturer instructions and stained with anti-MOK antibody bi-phosphorylated (pThr159+pTyr161, StressMarq, cat. SPC-1030), followed by donkey anti-rabbit IgG-Alexa Fluor 488 (Invitrogen, cat. A-21206).

Techniques: Staining, One-tailed Test, Western Blot, Two Tailed Test, Binding Assay, Control, Isolation

Journal: STAR Protocols

Article Title: Protocol for preparation of primary alveolar epithelial type I cells from mouse lungs

doi: 10.1016/j.xpro.2024.103484

Figure Lengend Snippet:

Article Snippet: Rabbit anti-mouse RAGE (1:100 dilution) , Boster , Cat# A03438; RRID: AB_3081636.

Techniques: Recombinant, Modification, Electron Microscopy, Software, Sterility

Journal: International Journal of Molecular Medicine

Article Title: High-mobility group box 1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway

doi: 10.3892/ijmm.2016.2537

Figure Lengend Snippet: High-mobility group box 1 protein (HMGB1) induces the activation of the receptor for advanced glycation end-products (RAGE)/extracellular signal-regulated kinase (ERK)1/2 pathway in 16HBE cells. The cells were stimulated with 400 ng/ml HMGB1 for the indicated periods of time. (A) The protein expression of Toll-like receptor (TLR)2, TLR4 and RAGE was detected by western blot analysis. HMGB1 only increased the protein expression level of RAGE. (B) The activation of 3 mitogen-activated protein kinase (MAPK) pathways [phosphorylated (p-)MAPK/total (t-)MAPK: p-ERK, t-ERK, p-p38 MAPK (p38), t-p38, p-c-Jun N-terminal kinase (JNK) and t-JNK] and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways ws detected by western blot analysis at the indicated time points. HMGB1 induced ERK1/2, Akt and JNK phosphorylation, but did not alter p38 phosphorylation. Representatives of the 3 independent experiments are shown.

Article Snippet: The antibodies utilized were as follows: rabbit anti-E-cadherin (sc-7870), rabbit anti-β-catenin (sc-7199), rabbit anti-occludin (sc-5562), and rabbit anti-claudin-1 (sc-28668) antibodies (from Santac Cruz Biotechnology); rabbit anti-phosphorylated (p-)p38 mitogen-activated protein kinase (MAPK) (p38) (4511), rabbit anti-total-p38 (8690), rabbit anti-p-ERK1/2 (4370), rabbit anti-total-ERK1/2 (4695), rabbit anti-p-c-Jun N-terminal kinase (JNK; 4668), rabbit anti-total-JNK (8690), rabbit anti-p-Akt (12178) and rabbit anti-total-Akt (4685) antibodies (all from Cell Signaling Technology); and mouse anti-RAGE antibody (MAB10040; from Merck Millipore, Darmstadt, Germany) After washing with TBS 0.1%/Tween-20 (Sigma-Aldrich), the membranes were incubated for 2 h at room temperature with a secondary antibody: an ECL-anti-rabbit or mouse IgG HRP-linked antibody from Cell Signaling Technology.

Techniques: Activation Assay, Expressing, Western Blot, Protein-Protein interactions, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: High-mobility group box 1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway

doi: 10.3892/ijmm.2016.2537

Figure Lengend Snippet: Blockade of the receptor for advanced glycation end-products (RAGE)/extracellular signal-regulated kinase (ERK)1/2 pathway attenuates high-mobility group box 1 protein (HMGB1)-induced epithelial barrier dysfunction in 16HBE cells. (A and B) To ascertain that the barrier dysfunction was mediated by RAGE, 16HBE cells were treated with or without anti-RAGE antibody (5 µ g/ml) for 1 h prior to stimulation with HMGB1 for 24 h (n=6 experiments) and then the transepithelial electrical resistance (TER) and FITC-dextran permeability were measured. To determine that barrier dysfunction depended on the activation of mitogen-activated protein kinases (MAPKs) or phosphatidylinositol 3-kinase (PI3K)/Akt signaling, TER and FITC-dextran permeability were measured (n=6 experiments) after the 16HBE cells were treated with or without the related signaling inhibitors: [(C and D) U0126 (ERK inhibitor, 10 µ M), (E and F) LY294002 (PI3K/Akt inhibitor, 25 µ M)], and (G and H) SP600125 (JNK inhibitor, 25 µ M)], prior to stimulatoin with HMGB1 for 24 h. The levels of TER were normalized to those prior to HMGB1 or medium only (control) stimulation, and the levels of the FITC-dextran permeability were normalized to the ratio between HMGB1 and medium only (control) stimulation, which was denoted by Pa/Pc. P-values were calculated using the ANOVA test. * P<0.05 vs. control; # P<0.05 vs. stimulation with 400 ng/ml HMGB1.

Article Snippet: The antibodies utilized were as follows: rabbit anti-E-cadherin (sc-7870), rabbit anti-β-catenin (sc-7199), rabbit anti-occludin (sc-5562), and rabbit anti-claudin-1 (sc-28668) antibodies (from Santac Cruz Biotechnology); rabbit anti-phosphorylated (p-)p38 mitogen-activated protein kinase (MAPK) (p38) (4511), rabbit anti-total-p38 (8690), rabbit anti-p-ERK1/2 (4370), rabbit anti-total-ERK1/2 (4695), rabbit anti-p-c-Jun N-terminal kinase (JNK; 4668), rabbit anti-total-JNK (8690), rabbit anti-p-Akt (12178) and rabbit anti-total-Akt (4685) antibodies (all from Cell Signaling Technology); and mouse anti-RAGE antibody (MAB10040; from Merck Millipore, Darmstadt, Germany) After washing with TBS 0.1%/Tween-20 (Sigma-Aldrich), the membranes were incubated for 2 h at room temperature with a secondary antibody: an ECL-anti-rabbit or mouse IgG HRP-linked antibody from Cell Signaling Technology.

Techniques: Permeability, Activation Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: High-mobility group box 1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway

doi: 10.3892/ijmm.2016.2537

Figure Lengend Snippet: Activation of the receptor for advanced glycation end-products (RAGE)/extracellular signal-regulated kinase (ERK)1/2 pathway by high-mobility group box 1 protein (HMGB1) is involved in the disruption of the epithelial intercellular contacts. The 16HBE cells were exposed to 400 ng/ml HMGB1 for the indicated periods of time. (A) 16HBE cells were treated with or without U0126 (ERK inhibitor, 10 µ M), prior to stimulation with HMGB1 for 6 h, phosphorylated (p-)ERK, and the total (t-)ERK were detected by western blot analysis. To confirm the association between RAGE and the activation of the ERK1/2 pathway, the 16HBE cells were treated with or without anti-RAGE antibody (5 µ g/ml) for 1 h prior to stimulation with HMGB1 for 6 h. The anti-RAGE antibody suppressed the HMGB1-induced p-ERK1/2; the response was verified by western blot analysis. (B) E-cadherin, β-catenin, occludin and claudin-1 were detected using western blot analysis. U0126 inhibited the HMGB1-mediated downregulation of the tight junction proteins, occludin and claudin-1. The ERK inhibitor, U0126, prevented the delocalization of (C) E-cadherin and (D β-catenin in response to HMGB1, verified by immunofluorescence staining. Representatives of 3 independent experiments are shown. * P<0.05 vs. HMGB1-stimulated group; scale bar, 50 mm.

Article Snippet: The antibodies utilized were as follows: rabbit anti-E-cadherin (sc-7870), rabbit anti-β-catenin (sc-7199), rabbit anti-occludin (sc-5562), and rabbit anti-claudin-1 (sc-28668) antibodies (from Santac Cruz Biotechnology); rabbit anti-phosphorylated (p-)p38 mitogen-activated protein kinase (MAPK) (p38) (4511), rabbit anti-total-p38 (8690), rabbit anti-p-ERK1/2 (4370), rabbit anti-total-ERK1/2 (4695), rabbit anti-p-c-Jun N-terminal kinase (JNK; 4668), rabbit anti-total-JNK (8690), rabbit anti-p-Akt (12178) and rabbit anti-total-Akt (4685) antibodies (all from Cell Signaling Technology); and mouse anti-RAGE antibody (MAB10040; from Merck Millipore, Darmstadt, Germany) After washing with TBS 0.1%/Tween-20 (Sigma-Aldrich), the membranes were incubated for 2 h at room temperature with a secondary antibody: an ECL-anti-rabbit or mouse IgG HRP-linked antibody from Cell Signaling Technology.

Techniques: Activation Assay, Disruption, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: HMGB1-RAGE Axis Makes No Contribution to Cardiac Remodeling Induced by Pressure-Overload

doi: 10.1371/journal.pone.0158514

Figure Lengend Snippet: Neonatal rat cardiomyocytes were exposed to recombinant HMGB1 (rHMGB1). (A) Effect of rHMGB1 on p-ERK1/2. (B) Cell viability was detected with MTT assay. (C) Cell proliferation detected with CCK8 assay. (D) Cell surface area in response to rHMGB1 in the absence/presence of anti-RAGE neutralizing antibody. Results are means ± SEM. n = 6 in each dose group in panel A-C, n = 50 cells in each group and * P <0.05 vs. 1 st bar in panel D.

Article Snippet: NRVCs were cultured for 1 day and treated with various concentrations of HMGB1 (Sigma-Aldrich Co.) and/or anti-RAGE neutralizing antibody (Merck Millipore Co).

Techniques: Recombinant, MTT Assay, CCK-8 Assay