Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-p38 antibody Clone D13E1 , Cell Signaling Technology , Cat# 8690s; RRID:AB_10999090.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Gel Extraction, Reverse Transcription, Quantitative RT-PCR, Plasmid Preparation, Isolation, Cell Culture, Immunoprecipitation, Gene Expression, CRISPR, Software, Sequencing, Modification

Journal: Frontiers in Immunology

Article Title: Streptococcus Suis Serotype 2 Stimulates Neutrophil Extracellular Traps Formation via Activation of p38 MAPK and ERK1/2

doi: 10.3389/fimmu.2018.02854

Figure Lengend Snippet: Role of the activation of p38 MAPK and ERK1/2 in SS2 and PMA induced NETs formation. (A) Neutrophils were infected with SS2 ZY05719 or treated with PMA for 2 h; p38 MAPK and ERK1/2 phosphorylation were determined with western blotting using specific antibody against phospho-p38 MAPK (p-p38), p38 MAPK (p38), phospho-ERK1/2 (p-ERK1/2), and ERK1/2. GAPDH was used as an internal control. (B) Neutrophils were pretreated with inhibitor of p38 MAPK (SB203580) and ERK1/2 (U0126), and then cells were incubated with SS2 ZY05719 or PMA for 3 h. Results were normalized to neutrophils without pretreatment and activation, and are depicted as the mean ±SEM ( n = 3) of three independent experiments; ** p < 0.01; *** p < 0.001. (C) Neutrophils were pretreated with inhibitor of p38 MAPK (SB203580) and ERK1/2 (U0126), and the cells were then incubated with either SS2 ZY05719 or PMA for 3 h. Immunofluorescence was performed using anti-neutrophil elastase (NE) antibody followed by goat anti-rabbit Alexa 568 antibody (red). DNA was stained with DAPI (blue). The results shown are representative of three independent experiments.

Article Snippet: Thereafter, the membranes were incubated with Phospho-p38 MAPK (Thr180/Thr182) (D3F9) XP ® Rabbit mAb, p38 MAPK (D13E1) XP® Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)XP ® Rabbit mAb, p44/42 MAPK (Erk1/2)(137F5) Rabbit mAb, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (1:1000, Cell Signaling Technology, Danvers, MA, USA), or anti-GAPDH Rabbit pAb (1:5000, CMCTAG, Milwaukee, WI, USA) at 4°C overnight.

Techniques: Activation Assay, Infection, Phospho-proteomics, Western Blot, Control, Incubation, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Streptococcus Suis Serotype 2 Stimulates Neutrophil Extracellular Traps Formation via Activation of p38 MAPK and ERK1/2

doi: 10.3389/fimmu.2018.02854

Figure Lengend Snippet: Western blotting analysis of phosphorylation of p38 MAPK and ERK1/2. (A) Neutrophils were pretreated with inhibitors of TLR4 signaling (TAK-242) and NADPH oxidase (DPI) for 30 min, and the cells were then incubated with SS2 ZY05719 and PMA for 2 h. Phosphorylation of p38 MAPK (p-p38) and ERK1/2 (p-ERK1/2) were determined with western blotting. (B) Neutrophils isolated form wild-type mice and TLR knockout mice were incubated with SS2 ZY05719 and Medium.

Article Snippet: Thereafter, the membranes were incubated with Phospho-p38 MAPK (Thr180/Thr182) (D3F9) XP ® Rabbit mAb, p38 MAPK (D13E1) XP® Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)XP ® Rabbit mAb, p44/42 MAPK (Erk1/2)(137F5) Rabbit mAb, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (1:1000, Cell Signaling Technology, Danvers, MA, USA), or anti-GAPDH Rabbit pAb (1:5000, CMCTAG, Milwaukee, WI, USA) at 4°C overnight.

Techniques: Western Blot, Phospho-proteomics, Incubation, Isolation, Knock-Out

Journal: Ecotoxicology and environmental safety

Article Title: SKI-606, a Src inhibitor, ameliorates benzene-induced hematotoxicity via blocking ROS/Src kinase-mediated p38 and Akt signaling pathways.

doi: 10.1016/j.ecoenv.2024.117223

Figure Lengend Snippet: Fig. 1. HQ induces hematotoxicity through ROS-dependent activation of Src kinase in HSPCs. (A) BMMNCs were treated with 0, 40, 60, 80 μM HQ for 2 h, and ROS production of CD34+ cells (HSPCs) were determined using DCF-DA assay with flow cytometry analysis. Representative images were shown representing three independent experiments. (B) HSPCs were treated with 0, 40, 60, 80 μM HQ for 24 h, and DNA damage was detected by immunofluorescence assay with anti-γH2AX antibody; scale bar = 20 μm. (C, D) STRING analysis based on GSE169037 indicated that Src was identified as a key kinase contributing to DNA damage. (E) HSPCs were treated with 0, 40, 60, 80 μM HQ for 24 h, and the phosphorylation levels of Src at Tyr416 or Tyr527, p38MAPK, and Akt were determined using western blot. (F) BMMNCs were treated with 5 mM NAC, 1 μM SKI-066 or 0.05 % DMSO for 2 h, and ROS production of CD34+ cells were determined using DCF-DA assay with flow cytometry analysis. Representative images were shown representing three independent experiments.

Article Snippet: Then, the protein was boiled and subjected to western blotting with primary antibodies against Src (Clone: 36D10), Phospho-Src (Tyr416) (Clone: D49G4), Phospho-Src (Tyr527), p38 MAPK (Clone: D13E1), Phospho-p38 MAPK (Clone: D3F9), Akt (Clone: C67E7), Phospho-Akt (Ser473) (Clone: D9E) (all from Cell Signaling Technology, Beverly, MA, USA), and GAPDH (Clone: 1E6D9; Proteintech, Chicago, IL, USA), respectively.

Techniques: Activation Assay, Flow Cytometry, Immunofluorescence, Phospho-proteomics, Western Blot

Journal: Ecotoxicology and environmental safety

Article Title: SKI-606, a Src inhibitor, ameliorates benzene-induced hematotoxicity via blocking ROS/Src kinase-mediated p38 and Akt signaling pathways.

doi: 10.1016/j.ecoenv.2024.117223

Figure Lengend Snippet: Fig. 2. SKI-606 alleviates HQ-induced hematotoxicity via blocking Src kinase-mediated p38 and Akt signaling pathways in HSPCs. (A, B) HSPCs were pre- treated with 1 μM SKI-606, 10 μM Akt agonist SC79 or 10 μM p38 inhibitor PD169316 for 15 min and then followed by exposure of 60 μM HQ for 24 h, and cells were harvested for analysis of the phosphorylation levels of Src at Tyr416, p38 and Akt by western blot. (C) The colony formation of HSPCs treated with or without 60 μM HQ or 1 μM SKI-606 was evaluated. Representative images (left label) and statistical analysis (right label) were shown, scale bar = 100 & 200 μm. (D) The apoptosis of HSPCs treated with or without 60 μM HQ or 1 μM SKI-606 for 24 h was detected by flow cytometry. Representative images (left label) and statistical analysis (right label) were shown. Data are expressed as mean ± SD. ***P < 0.001, vs vehicle group.

Article Snippet: Then, the protein was boiled and subjected to western blotting with primary antibodies against Src (Clone: 36D10), Phospho-Src (Tyr416) (Clone: D49G4), Phospho-Src (Tyr527), p38 MAPK (Clone: D13E1), Phospho-p38 MAPK (Clone: D3F9), Akt (Clone: C67E7), Phospho-Akt (Ser473) (Clone: D9E) (all from Cell Signaling Technology, Beverly, MA, USA), and GAPDH (Clone: 1E6D9; Proteintech, Chicago, IL, USA), respectively.

Techniques: Blocking Assay, Protein-Protein interactions, Phospho-proteomics, Western Blot, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Danshen ( Salvia miltiorrhiza ) restricts MD2/TLR4‐MyD88 complex formation and signalling in acute myocardial infarction‐induced heart failure

doi: 10.1111/jcmm.15688

Figure Lengend Snippet: Primary antibodies used in Western blot experiment

Article Snippet: TLR4 MyD88 TRAF6 IκBα p‐IκBα MAPK p‐MAPK NF‐κB p‐NF‐κB GAPDH , TLR4 Antibody, 19811‐1‐AP, Proteintech Anti‐MyD88, ab2064, Abcam Anti‐TRAF6, ab227560, Abcam Ant‐IKBα, ab32518, Abcam Phospho‐IκBα (Ser32), 2859, CST p38 MAPK, 2387, CST Phospho‐p38 MAPK (Thr180/Tyr182), 9215, CST Anti‐NF‐κB p65, ab16502, Abcam Anti‐NF‐κB p65 (phospho S529), ab97726, Abcam Anti‐GAPDH, ab8246, Abcam , 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:2000.

Techniques: Western Blot, Concentration Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Danshen ( Salvia miltiorrhiza ) restricts MD2/TLR4‐MyD88 complex formation and signalling in acute myocardial infarction‐induced heart failure

doi: 10.1111/jcmm.15688

Figure Lengend Snippet: Danshen is shown to regulate critical signalling molecules of TLR4 signalling pathway. (A) Western blots images and analysis of TLR4, MyD88, TRAF6, p‐IκB, p‐p38 MAPK and p‐NF‐κB. N = 3. (B) IHC and quantitative result of TLR4 in different treatment groups, scale bar = 20 μm, N = 5. * P < .05, ** P < .01, ** P < .01

Article Snippet: TLR4 MyD88 TRAF6 IκBα p‐IκBα MAPK p‐MAPK NF‐κB p‐NF‐κB GAPDH , TLR4 Antibody, 19811‐1‐AP, Proteintech Anti‐MyD88, ab2064, Abcam Anti‐TRAF6, ab227560, Abcam Ant‐IKBα, ab32518, Abcam Phospho‐IκBα (Ser32), 2859, CST p38 MAPK, 2387, CST Phospho‐p38 MAPK (Thr180/Tyr182), 9215, CST Anti‐NF‐κB p65, ab16502, Abcam Anti‐NF‐κB p65 (phospho S529), ab97726, Abcam Anti‐GAPDH, ab8246, Abcam , 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:2000.

Techniques: Western Blot

Journal: Cell reports

Article Title: PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity

doi: 10.1016/j.celrep.2020.108258

Figure Lengend Snippet: (A) Diagram showing the signaling pathway of chemokine receptors, including CCR7 and CXCR4. Colored text indicates steps of the pathway interrogated, with red showing steps disrupted by loss of PD-L1 signaling and blue showing steps of the pathway without significant changes. (B) Quantification of IP-1 concentration in WT and Pdl1 CyMt BMDCs following 30 min of CCL21 (1 μg/mL) treatment. (C) Western blot image using antibodies against ERK1/2, pERK1/2, p38, p-p38, and GAPDH at 0, 1, or 5 min following CCL21 (1 μg/mL) addition to BMDC media. The experiment was repeated twice with similar results. (D) Quantification of (C), showing fold change over no chemokine. (E) Representative flow plots of F-actin following 15 s of CCL21 (1 μg/mL) treatment in BMDCs. (F) Quantification of (E) showing fold change over no chemokine at 15s, 60 s, and 5 min after CCL21. n = 3 technical replicates per group. The data shown are combined from three independent experiments. (G) Representative flow plots of F-actin following 15 s of S1P (500 nM) treatment in BMDCs. (H) Quantification of (G) showing fold change over no chemokine. n = 3 technical replicates per group. The data shown are combined from two independent experiments. Statistical analysis was done using a one-way ANOVA (B and F) or an unpaired Student’s t test (D and H). *p < 0.05; **p < 0.01; ****p < 0.0001; n.s., p > 0.05. Error bars indicate standard error of the mean. See also .

Article Snippet: Antibodies: total ERK1/2 (Biolegend W15133B), phospho-ERK1/2 (Cell Signaling 9109), total P38 (Cell Signaling D13E1), phoshpo-P38 (Cell Signaling D3F9), total STAT3 (Cell Signaling 79D7), phospho-STAT3 (Cell Signaling 9145), phospho-AKT (Cell Signaling, Cat#9271S), phospho-AKT (Cell Signaling, Cat#4060S), and GAPDH-HRP (Invitrogen MA5-15738-HRP).

Techniques: Concentration Assay, Western Blot

Journal: Cell reports

Article Title: PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity

doi: 10.1016/j.celrep.2020.108258

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies: total ERK1/2 (Biolegend W15133B), phospho-ERK1/2 (Cell Signaling 9109), total P38 (Cell Signaling D13E1), phoshpo-P38 (Cell Signaling D3F9), total STAT3 (Cell Signaling 79D7), phospho-STAT3 (Cell Signaling 9145), phospho-AKT (Cell Signaling, Cat#9271S), phospho-AKT (Cell Signaling, Cat#4060S), and GAPDH-HRP (Invitrogen MA5-15738-HRP).

Techniques: Recombinant, Cell Isolation, Fluorescence, Software