Journal: Experimental and Therapeutic Medicine

Article Title: Proliferation, apoptosis and invasion of human lung cancer cells are associated with NFATc1

doi: 10.3892/etm.2022.11748

Figure Lengend Snippet: Knockdown of NFATc1 expression induces cell apoptosis whilst inhibiting the MAPK and epithelial-to-mesenchymal transition signaling pathways in lung cancer cells. (A) Protein expression levels of Bax, cleaved caspase-3, CDK4 and c-Myc in both cell lines were examined by western blot analysis. GAPDH was used as the loading control. (B) Protein expression levels of E-cadherin and N-cadherin were examined by western blotting. GAPDH was used as the loading control. (C) Protein expression levels of ERK and p38, along with their corresponding phosphorylation levels were detected by western blotting. GAPDH was used as the loading control. * P<0.05, ** P<0.01 and *** P<0.001 vs. shCtrl. Ctrl, control; NFATc1, nuclear factor of activated T cells c1; p-, phosphorylated; sh, short hairpin RNA.

Article Snippet: The blots were blocked in 5% skimmed milk for 1 h at room temperature, followed by incubation with primary antibodies against ERK (cat. no. 9107; 1:2,000; Cell Signaling Technology, Inc.), E-cadherin (cat. no. 14472; 1:500; Cell Signaling Technology, Inc.), phosphorylated (p)-ERK (cat. no. 4376; 1:1,000; Cell Signaling Technology, Inc.), N-cadherin (cat. no. ab18203; 1:500; Abcam), Bax (cat. no. ab32503; 1:2,000; Abcam), p38 (cat. no. 8690; 1:3,000; Cell Signaling Technology, Inc.), cleaved caspase-3 (cat. no. 9664; 1:500; Cell Signaling Technology, Inc.), p-p38 (cat. no. 4631; 1:500; Cell Signaling Technology, Inc.), c-Myc (cat. no. ab32072; 1:1,000; Abcam), CDK4 (cat. no. ab137675; 1:1,000; Abcam), NFAT2 (cat. no. ab2796; 1:1,000; Abcam) and GAPDH (cat. no. ab37168; 1:5,000; Abcam) at 4 ̊C with gentle shaking overnight.

Techniques: Knockdown, Expressing, Protein-Protein interactions, Western Blot, Control, Phospho-proteomics, shRNA

Journal: Biological & pharmaceutical bulletin

Article Title: CoCl 2 Decreases EC-SOD Expression through Histone Deacetylation in COS7 Cells.

doi: 10.1248/bpb.b16-00551

Figure Lengend Snippet: Fig. 2. Involvement of p38-MAPK in CoCl2-Elicited Histone Deacety- lation in COS7 Cells

Article Snippet: After transferring electrophoretically onto polyvinylidene difluoride (PVDF) membranes, the membranes were incubated with anti-acetyl-histone H3 (06-599, Millipore: 1 : 2000), anti-acetyl-histone H4 (06-598, Millipore: 1 : 2000), or anti-phospho-p38 MAPK (#9215, Cell Signaling: 1 : 1000) overnight.

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: CoCl 2 Decreases EC-SOD Expression through Histone Deacetylation in COS7 Cells.

doi: 10.1248/bpb.b16-00551

Figure Lengend Snippet: Fig. 4. Luteolin Suppresses CoCl2-Elicited Decreases in EC-SOD by Inhibiting ROS-p38-MAPK Signaling in COS7 Cells

Article Snippet: After transferring electrophoretically onto polyvinylidene difluoride (PVDF) membranes, the membranes were incubated with anti-acetyl-histone H3 (06-599, Millipore: 1 : 2000), anti-acetyl-histone H4 (06-598, Millipore: 1 : 2000), or anti-phospho-p38 MAPK (#9215, Cell Signaling: 1 : 1000) overnight.

Techniques:

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: Cellular energy stress suppresses PGN-induced NOD1 signaling (A) Mouse BMDM cells were treated with or without glucose for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (B) Mouse BMDM cells were treated with 2-DG (25 mM) in glucose-free medium for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (C) Mouse BMDM cells were treated with metformin (2 mM) for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (D) Mouse iBMDM cells were treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (E) Mouse iBMDM cells were treated with 2-DG (25 mM) in glucose-free medium and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (F) Mouse iBMDM cells were pre-treated with DMSO or Compound C (5 μM) and treated with or without glucose for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (G) Mouse iBMDM cells were pre-treated with DMSO or Compound C (5 μM) and then treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (H) Mouse iBMDM cells were treated with MK-8722 (2 μM) for 8 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (I) HEK293T cells were treated with or without glucose for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (J) HEK-293T cells expressing FLAG-NOD1 were treated with or without glucose for 6h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (K) HEK293T cells were pre-treated with DMSO or Compound C (5 μM) and then treated with glucose starvation for 6h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (L) HEK-293T cells expressing FLAG-NOD1 were pre-treated with DMSO or Compound C (5 μM) and treated with or without glucose for 6h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies.

Article Snippet: Rabbit-Anti-Phospho-p38 MAPK (Thr180/Tyr182) , Cell Signaling Technology , Cat# 4511; RRID: AB_2139682.

Techniques: Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, Expressing, Membrane

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: AMPK-mediated ZDHHC5 phosphorylation inhibits NOD1 activation (A) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were transfected to express FLAG-NOD1, then treated with metformin (5 mM) and labeled with alk-C16 for 6 h. NOD1 palmitoylation was detected by click chemistry reaction. (B) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were treated with metformin (5 mM) for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (C) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were treated with or without glucose for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (D) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were transfected to express FLAG-NOD1, and treated with metformin (5 mM) for 6 h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (E) BMDMs were generated from Zdhhc5 −/− mice, and were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted BMDM cells were treated with metformin (2 mM) for 6 h, followed by stimulation with C12-iE-DAP (1 μg/mL) for 30 min. p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (F) ZDHHC5-knockdown iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDM cells were treated with metformin (2 mM) for 6 h, followed by stimulation with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (G) ZDHHC5-knockdown iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDMs cells were treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test.

Article Snippet: Rabbit-Anti-Phospho-p38 MAPK (Thr180/Tyr182) , Cell Signaling Technology , Cat# 4511; RRID: AB_2139682.

Techniques: Phospho-proteomics, Activation Assay, Knock-Out, Mutagenesis, Transfection, Labeling, Fluorescence, Membrane, Generated, Transduction, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Brain, behavior, and immunity

Article Title: The role of the Toll like receptor 4 signaling in sex-specific persistency of depression-like behavior in response to chronic stress

doi: 10.1016/j.bbi.2023.10.006

Figure Lengend Snippet: (A) Experimental outline. Unstressed mice that were not exposed to chronic stress and age-matched (upper panel) and stressed mice that were exposed to chronic stress exposure for 28 days, followed by 28 days of post-stress period. (B and C) Bar graphs showing total immobility time in female (red bar) and male (blue bar) stressed mice compared to unstressed mice at D28 (B) and at D56(C). Statistical analyses were performed using Two-way ANOVA (stress effect, p < 0.0001; sex effect, p = 0.2065; interaction effect, p = 0.0030; * p < 0.05, **** p < 0.001 compared to unstressed male mice; #### p < 0.0001, compared to unstressed female mice, + p < 0.05, +++ p < 0.001, compared to stressed male mice, n = 7–9 mice for each group). (D) A graph showing depression-like behavior over the entire experimental, with female mice shown in red and male mice shown in blue. (E-N) Microglia activation and morphological analysis in female and male mice at D56. (E and J) Representative immunofluorescence images of IBA-1 + (red) and CD68 + (yellow) cells in female (E.1 and E.2) and male (J.1 and J.2) mice. (F and K) Bar graphs showing number of IBA-1 + cells in female (F) and male (K) mice. (G and L) Bar graphs showing quantification of CD68 + area per IBA-1 + microglia in female (G) and male (L) mice. (E and J) Representative images of three-dimensional (3D) reconstruction in female (E.3, E.4, E.5 and E.6) male (J.3, J.4, J.5 and J.6) mice. The outlined with a white box is magnified. (H-N) Sholl analysis based on 3D reconstruction in female and male mice. Bar graph showing soma size per microglia in female (H) and male (M) mice. Line graphs showing quantification of number of intersection between each circle (green, E5, E6, J5 and J6) in female (I) and male (N) mice. Statistical analyses were performed using t -test (* p < 0.05, compared to unstressed female mice). Scale bars indicate 20 (E and J.1–2) and 10 (E and J.3–6) um. Each dot represents an individual mouse. Results are expressed as the mean ± SEM.

Article Snippet: The membrane was then blocked for 1 h at room temperature and treated overnight with primary antibodies specific to p-NF-kB p65 (3033 s, Abcam), NF-kB p65 (51–0500, Thermo Fisher Scientific), caspase-1 (20B-0042, adipogen, CA, USA), IL-1β (6243S, Cell signaling, MA, USA), and α-tubulin (T9026, Sigma) at 4 °C.

Techniques: Activation Assay, Immunofluorescence

Journal: Brain, behavior, and immunity

Article Title: The role of the Toll like receptor 4 signaling in sex-specific persistency of depression-like behavior in response to chronic stress

doi: 10.1016/j.bbi.2023.10.006

Figure Lengend Snippet: (A) The experimental outline in Cx3Cr1 CreEr ; Tlr4 fl double transgenic mice. Unstressed female mice that were not exposed to chronic stress and age-matched and stressed female mice that were exposed to chronic stress for 28 days, followed by 28 days of post-stress period, and then treated with vehicle or tamoxifen for an additional 12 days. (B-D) Bar graphs showing total immobility time at D28 (B), D56 (C) and D68 (D). (E-G) Representative western blot of p-NF-KB 65 (E, upper panel), NF-kB p65 (E, upper panel), caspase-1 (F, upper panel) and IL-1β (G, upper panel) and bar graphs showing relative quantification of p-NF-KB p65 (E, bottom left panel) normalized to NF-kB p65, NF-kB p65 (E, bottom right panel), cleaved caspase-1 (F, bottom panel) and IL-1β (G, bottom panel) levels normalized to α-tubulin. (H) Bar graph showing concentration of IL-1β in PFC lysates. Statistical analyses were performed using t -test or One-way ANOVA (* p < 0.05, ** p < 0.01, **** p < 0.0001 compared to female unstressed mice; # p < 0.05, ## p < 0.01, ### p < 0.001, compared to female stressed mice, n = 4–5 mice for each group). Each dot represents an individual mouse. Results are expressed as the mean ± SEM.

Article Snippet: The membrane was then blocked for 1 h at room temperature and treated overnight with primary antibodies specific to p-NF-kB p65 (3033 s, Abcam), NF-kB p65 (51–0500, Thermo Fisher Scientific), caspase-1 (20B-0042, adipogen, CA, USA), IL-1β (6243S, Cell signaling, MA, USA), and α-tubulin (T9026, Sigma) at 4 °C.

Techniques: Transgenic Assay, Western Blot, Quantitative Proteomics, Concentration Assay

Journal: Brain, behavior, and immunity

Article Title: The role of the Toll like receptor 4 signaling in sex-specific persistency of depression-like behavior in response to chronic stress

doi: 10.1016/j.bbi.2023.10.006

Figure Lengend Snippet: (A) Schematic showing TRAP-sequencing. (B) Selected Gene ontology (GO) annotations enriched in GFP-bound (green, upper panel) and GFP-unbound group (gray, lower panel).The x-axis represents - log10 (p-value), with dotted line representing a p -value of 0.05. (C-D) Volcano plots in female (C) and male (D) mice. The x-axis represents the log 2 conversion of the fold change (log 2 FC) values, and the y-axis represents the corrected significance level after base log10 conversion ( p value). Red and blue dots in the volcano plot indicate all DEGs that were found to differ significantly (absolute value of log 2 FC > 1, * p < 0.05). (E) Heat map with hierarchical clustering distances shows the variation in the expression levels (VST-scored log 2 RPKM) between female stressed and unstressed mice. (F) A bar graph showing selected GO annotations enriched in female (red) and male (blue) mice. The x-axis represents -log10 ( p -value), with dotted line representing a p -value of 0.05. (G) A bar graph showing log 2 FC of significant DEGs in female stress (red) and male stress (blue) mice using a cut-off of p < 0.05.

Article Snippet: The membrane was then blocked for 1 h at room temperature and treated overnight with primary antibodies specific to p-NF-kB p65 (3033 s, Abcam), NF-kB p65 (51–0500, Thermo Fisher Scientific), caspase-1 (20B-0042, adipogen, CA, USA), IL-1β (6243S, Cell signaling, MA, USA), and α-tubulin (T9026, Sigma) at 4 °C.

Techniques: Sequencing, Expressing

Journal: Cell

Article Title: Ribosome collisions trigger general stress responses to regulate cell fate

doi: 10.1016/j.cell.2020.06.006

Figure Lengend Snippet: (A) Immunoblots for phosphorylation of eIF2α and mTOR in MCF10A cells treated with ANS (0.1–100 mg/L, 0.5 h). Total eIF2α and β-actin as loading controls. (B) eIF2α phosphorylation induced by intermediate doses of ANS (0.5 mg/L, 0.5 h) in MCF10A cells pretreated with GCN2 inhibitor (A-92), PERK inhibitor (GSK 2606414), or p38 inhibitor (BIRB 796). (C) eIF2α phosphorylation induced by ANS in WT or ZAK KO MCF10A cells. (D) eIF2α phosphorylation in WT, ZAK KO, or ZAK KO MCF10A complemented with ZAKα, ZAKα -K45M, or ZAKβ under ANS treatment (0.5 mg/L, 0.5 h). (E) Polysome profiles from DSP-crosslinked WT and ZAK KO MCF10A cells. Fractions were analyzed by immunoblotting with indicated antibodies.

Article Snippet: Antibodies for phospho-p38 (Thr180/Tyr182, 9211), p38 (9212), phospho-JNK (Thr183/Tyr185, 4668), β-actin (51255), phospho-mTOR (Ser2448, 2971) and eIF2α (9722S) were from Cell Signaling Technology.

Techniques: Western Blot, Phospho-proteomics

Journal: Disease Markers

Article Title: Aldolase A Promotes Colorectal Cancer Progression through Targeting COPS6 and Regulating MAPK Signaling Pathway

doi: 10.1155/2023/1702125

Figure Lengend Snippet: Graphical abstract. By binding to and targeting COPS6, aberrantly expressed ALDOA promoted the EMT process and activated the ERK1/2 and P38 signaling pathways, ultimately accelerating CRC cell proliferation and metastasis.

Article Snippet: The following antibodies were used: ALDOA (mouse monoclonal; cat. no. sc-390733; 1 : 1000 dilution; Santa Cruz, CA, USA), HRP-conjugated DYKDDDDK Tag (monoclonal; cat. no. HRP-66008; 1 : 5000 dilution; Proteintech Group), ALDOB (rabbit polyclonal; cat. no. 18065-1-AP; 1 : 1000 dilution; Proteintech Group), ALDOC (rabbit polyclonal; cat. no. 14884-1-AP; 1 : 1000 dilution; Proteintech Group), E-cadherin (rabbit monoclonal; cat. no. 3195; 1 : 1000 dilution; Cell Signaling Technology), N-cadherin (rabbit monoclonal; cat. no. 13116; 1 : 1000 dilution; Cell Signaling Technology), vimentin (rabbit monoclonal; cat. no. 5741; 1 : 1000 dilution; Cell Signaling Technology), p38 (rabbit monoclonal; cat. no. 8690; 1 : 1000 dilution; Cell Signaling Technology), p-p38 (rabbit monoclonal; cat. no. 8632; 1 : 1000 dilution; Cell Signaling Technology), ERK1/2 (rabbit monoclonal; cat. no. 4695; 1 : 1000 dilution; Cell Signaling Technology), p-ERK1/2 (rabbit monoclonal; cat. no. 4376; 1 : 1000 dilution; Cell Signaling Technology), ACTB (rabbit monoclonal; cat. no. AC038; 1 : 10000 dilution; ABclonal Technology, Wuhan, China), GAPDH (rabbit monoclonal; cat. no. 60004-1-Ig; 1 : 10000 dilution; Proteintech Group), lamin B1 (rabbit polyclonal; cat. no. 12987-1-AP; 1 : 2000 dilution; Proteintech Group), PKM (rabbit polyclonal; cat. no. 10078-2-AP; 1 : 1000 dilution; Proteintech Group), HSP90AB (rabbit polyclonal; cat. no. RK05737; 1 : 1000 dilution; ABclonal Technology), CSN6 (mouse monoclonal; cat. no. sc-393023; 1 : 1000 dilution; Santa Cruz, CA, USA), caspase-3 (rabbit monoclonal; cat. no. 9662; 1 : 1000 dilution; Cell Signaling Technology), and cleaved caspase-3 (rabbit monoclonal; cat. no. 9661; 1 : 1000 dilution; Cell Signaling Technology).

Techniques: Binding Assay, Protein-Protein interactions

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p < 0.05 compared to the negative control group.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Inhibition, Western Blot, Phospho-proteomics, In Vitro, Migration, Negative Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon up-regulates p38β expression in hepatic cells. Pokemon was delivered by expression plasmid pcDNA3.1(−)-Pokemon with pcDNA3.1(−) as a negative control. Pokemon silencing was triggered by si-RNA. ( a ) Targeted expression or silencing of Pokemon in HepG2 cells. Cells were collected at 60 h after transfection or silencing (Left panel). And the quantification of western blot data was displayed on the Right panel; ( b ) Real-time quantitative polymerase chain reaction (qPCR) at 48 h after transfection in HepG2 cells. Upper panel: Ectopic expression of Pokemon; lower panel: Silencing of Pokemon; ( c ) Ectopic expression of Pokemon in HL7702 cells. Upper panel: Western blot in which cells were lysed and total proteins were collected at 36, 48, 60 and 72 h, respectively. Lower panel: Real-time qPCR at 48 and 60 h after transfection; ( d ) Silencing of Pokemon in BEL7404 cells. Upper panel: Western blot; lower panel: Real-time qPCR. * p < 0.05 compared to the negative control. N.C. means negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Negative Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon stimulates p38β promoter activity. ( a ) ChIP assays in HepG2 and BEL7404 cells. Protein-DNA complexes are immunoprecipitated either with anti-Pokemon antibody or anti-IgG as negative control, followed by PCR with primers specific to p38β promoter sequence and agarose-gel electrophoresis for visualization. Total lysates were used as the input samples and positive control; ( b ) Dual luciferase reporter assay. Luciferase activities were normalized to Renilla activity. Y axis stands for the relative fold changes of activity as the pcDNA3.1(−)-Pokemon plasmid increases. * p < 0.05 compared to the negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Activity Assay, Immunoprecipitation, Negative Control, Sequencing, Agarose Gel Electrophoresis, Positive Control, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Arthritis and rheumatism

Article Title: Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies.

doi: 10.1002/art.21009

Figure Lengend Snippet: Figure 4. Effects of IgG aPL on A, the phosphorylation of p38 MAPK and B, the induction of inducible nitric oxide synthase (iNOS). HUVECs were grown to confluence and treated for A, 120 minutes (p38 MAPK experiments; IgG aPL samples 1–6 and IgG NHS samples 1 and 2) or B, 60 minutes (iNOS experiments; IgG aPL samples 1–4 and IgG NHS sample 1) with 100 g/ml of IgG aPL, 100 g/ml of IgG NHS, or 2 g/ml of LPS. Western blot analyses to determine the phosphorylation of p38 MAPK and the expression of iNOS were performed as described in Materials and Methods. Actin was used as the housekeeping gene. Bands were quantified by densitometric analysis as described in Materials and Methods. See Figure 1 for other definitions.

Article Snippet: The membranes were blocked in 5% dry milk, incubated with a 1:2,000 dilution of mouse monoclonal anti-human phosphorylated p38 MAPK (Thr180/Tyr182) (antibody 28b10; Cell Signaling Technology, Beverly, MA) or with rabbit polyclonal anti-human NOS2 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by a 1-hour incubation in a 1:2,000 dilution of horseradish peroxidase– conjugated goat anti-mouse or goat anti-rabbit IgG.

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Arthritis and rheumatism

Article Title: Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies.

doi: 10.1002/art.21009

Figure Lengend Snippet: Figure 6. Proposed intracellular events in antiphospholipid antibody (aPL)–activated endothelial cells (ECs). The diagram shows some intracellular pathways mediated by aPL in ECs and the effects of specific inhibitors. ? uncertain; activation; – inhibition; P phosphorylation; broken arrows unconfirmed pathway; solid ar- rows confirmed pathway; IL-1 interleukin-1; p38 MAPK p38 mitogen-activated protein kinase; iNOS inducible nitric oxide synthase; NO nitric oxide; ICAM-1 intercellular adhesion mole- cule 1; VCAM-1 vascular cell adhesion molecule 1.

Article Snippet: The membranes were blocked in 5% dry milk, incubated with a 1:2,000 dilution of mouse monoclonal anti-human phosphorylated p38 MAPK (Thr180/Tyr182) (antibody 28b10; Cell Signaling Technology, Beverly, MA) or with rabbit polyclonal anti-human NOS2 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by a 1-hour incubation in a 1:2,000 dilution of horseradish peroxidase– conjugated goat anti-mouse or goat anti-rabbit IgG.

Techniques: Activation Assay, Inhibition, Phospho-proteomics