Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of the MAP kinase and NF-κB signaling pathways in mediating ECFP/Ang II-induced NHE3 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that in wild-type mPCT cells, ECFP/Ang II stimulated NHE3 expression significantly, and the response was attenuated by the MEK1/MEK2 kinase inhibitor U0126 and the NF-κB activation inhibitor Ro 106–9920, respectively. However, the MEK inhibitor PD 980659 and the p38 MAP kinase inhibitor SB202196 failed to attenuate the effect of ECFP/Ang II on NHE3 expression. Panel ( B ) shows that in Agtr1a -/- mPCT cells, ECFP/Ang II failed to stimulate NHE3 expression, and the inhibitors of the MAP kinases and NF-κB signaling pathways had no significant effects on NHE3 expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced Na + /HCO 3 - cotransporter expression in wild-type mPCT cells. Panel ( A ) shows that ECFP/Ang II significantly increased Na + /HCO 3 - expression, and the response was attenuated by losartan but not by PD123319, suggesting a dominant role of AT 1 receptors in mPCT cells. Panel ( B ) shows that the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659 attenuated the effects of ECFP/Ang II on expression, but the p38 MAP kinase inhibitor SB202196 had no effect on Na + /HCO 3 - expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that ECFP/Ang II increased NF-κB, p65 expression in wild-type mPCT cells, and the response was attenuated by both losartan and PD123319, supporting an important role of AT 1 and AT 2 receptors in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. Panel ( B ) shows that ECFP/Ang II alone had no significant effect on NF-κB, p65 expression in Agtr1a -/- mPCT cells, but both losartan and PD123319 potentiated this response. Panel ( C ) shows that in wild-type mPCT cells, the effect of ECFP/Ang II on NF-κB, p65 expression was attenuated by the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659, respectively. However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. ** p < 0.01 vs. control WT or Agtr1a -/- mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II, or Agtr1a -/- mPCT cells transfected with ECFP/ANG II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ reduces cSMAC formation and proximal BCR signaling at the immune synapse. Raji D1.3 B cells were transfected with either control siRNA or GMFγ siRNA and added to COS-7 APCs expressing the mHEL-HaloTag Ag (magenta). The cells were fixed at the indicated times and stained with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79, cyan). The B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images from one of five independent experiments. Scale bars: 5 μm. (B) The total fluorescence intensity of the mHEL-HaloTag Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each B cell and the median values were calculated for each time point. Each symbol on the graph represents the median value for the GMFγ knockdown cells, expressed as a percent of the median value for the control siRNA-transfected cells for the same time point in the same experiment. The differently shaped symbols represent five independent experiments. Paired t -tests were used to calculate p -values. (C) The percent of cells that had formed a cSMAC, defined as > 90% of the total Ag fluorescence intensity being contained in one or two clusters, is graphed. The different symbols represent independent experiments. Paired t -tests were used to calculate p -values. (D) The total fluorescence intensity of pCD79 that was present in clusters at the B cell-APC contact site was quantified for each B cell. The left panel shows representative data from one experiment. Each dot is one cell. n > 31 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. The Mann-Whitney U -test was used to calculate p -values. The right panel shows the results from five independent experiments, presented as in (B) , with n > 30 cells per condition in each experiment. Each symbol represents a single experiment in which the median pCD79 fluorescence intensity for GMFγ-depleted cells is expressed as a percent of the corresponding median value for the control cells. Paired t -tests were used to calculate p -values. (E) For each B cell represented in (D) , the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of the clustered mHEL-HaloTag Ag. The median (blue line) and interquartile ranges (black box) are shown. The data are presented as in (B , D) . **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p ≤ 0.05; ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Staining, Microscopy, Fluorescence, Knockdown, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ does not impair CD79 phosphorylation induced by soluble anti-Ig antibodies. Cells were transfected with either control siRNA or GMFγ siRNA. (A) Raji D1.3 B cells were stimulated with 20 μg/mL goat anti-mouse IgM to initiate signaling through the D1.3 BCR. (B) Ramos B cells were stimulated with donkey anti-human IgM. Cell lysates were analyzed by immunoblotting with antibodies that recognize the phosphorylated CD79a ITAM (pCD79a), actin (loading control), or GMFγ. The left panels show a representative experiment. The right panels show results from 4 independent experiments. For each sample, the pCD79a band intensity was divided by the corresponding actin band intensity (loading control). The resulting ratios were normalized to that for the 0 min control siRNA cell sample (defined as 1.0) in the same experiment. On the graph, each of the four experiments is indicated by a different symbol. The bars show the mean ± SEM. Paired t -tests were used to calculate p -values. ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Phospho-proteomics, Transfection, Control, Western Blot

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: 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: 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