Journal: Oncogene

Article Title: The fungicide ciclopirox inhibits lymphatic endothelial cell tube formation by suppressing VEGFR-3-mediated ERK signaling pathway.

doi: 10.1038/onc.2010.590

Figure Lengend Snippet: Figure 5 CPX inhibits VEGFR-3-mediated ERK1/2 pathway. (a, b) CPX inhibited phosphorylation of ERK1/2, but not Akt, JNK and p38 mitogen-activated protein kinase, in LECs in a concentration- and time-dependent manner. LECs treated with CPX (0–5 mM) for 24 h (a) or CPX (5 mM) for 0–24 h (b) were harvested and subjected to western blot analysis with indicated antibodies. b-Tubulin was used as a loading control. (c) Overexpression of VEGFR-3 conferred resistance to CPX inhibition of ERK1/2 phosphorylation in LECs. LEC/V (control) and LEC/VEGFR-3 were treated with or without CPX (5 mM) for 24 h, followed by western blotting with indicated antibodies. (d) Downregulation of VEGFR-3 mimicked the effect of CPX, inhibiting phosphorylation of ERK1/2 in LECs. LECs, infected with lentiviral shRNAs to VEGFR-3 and GFP (control), respectively, were treated with CPX (5 mM) for 24 h, followed by western blotting with indicated antibodies.

Article Snippet: The primary antibodies used included antibodies to VEGFR-3, glyceraldehyde 3-phosphate dehydrogenase, Akt, ERK2, JNK1, phospho-JNK (Thr183/ Tyr185), p38, phospho-p38 (Thr180/Tyr182), MKK1, Flag (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphoERK1/2 (Thr202/Tyr204), phospho-Akt (Ser473) (Cell Signaling, Beverly, MA, USA) and b-tubulin (Sigma).

Techniques: Phospho-proteomics, Concentration Assay, Western Blot, Control, Over Expression, Inhibition, Infection

Journal: Oncology reports

Article Title: Resveratrol attenuates matrix metalloproteinase-9 and -2-regulated differentiation of HTB94 chondrosarcoma cells through the p38 kinase and JNK pathways.

doi: 10.3892/or.2014.3192

Figure Lengend Snippet: Figure 5. Effects of resveratrol on MMP-stimulated differentiation of HTB94 cells via the p38 kinase and JNK pathways. HTB94 cells were untreated (control) or treated with 50 µM of resveratrol for the indicated time periods or with the indicated various concentrations of resveratrol for 24 h. (A) Expression of pp38, p38, pJNK, JNK and actin was detected using western blot analysis. HTB94 cells were untreated (control) or treated with resveratrol in the absence or presence of 20 µM SB203580 (SB) or 20 µM SP600125 (SP) for 24 h. (B) Cell proliferation was measured using MTT assay. (C) Activation of MMP-2 and MMP-9 was detected using gelatin zymography assay, and expression of type Ⅱ collagen, SOX-9, pp38, p38, pJNK, JNK and actin was detected using western blot analysis. (D) Accumulation of sulfated proteoglycan was determined by Alcain blue staining. Statistically significant differences were noted between the control and the treatment groups. These data are the results of a typical experiment. *P<0.05 compared to the control.

Article Snippet: Antibodies specific for MMP-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP-9 (Santa Cruz Biotechnology), type II collagen (Santa Cruz Biotechnology), SOX-9 (Santa Cruz Biotechnology), phosphorylated (p)p38 (Cell Signaling Technology, Danvers, MA, USA), p38 (Santa Cruz Biotechnology), pJNK (Cell Signaling Technology), JNK (Santa Cruz Biotechnology) and actin (Santa Cruz Biotechnology) were used for the experiments.

Techniques: Control, Expressing, Western Blot, MTT Assay, Activation Assay, Zymography Assay, Staining

Journal: Oncology reports

Article Title: Resveratrol attenuates matrix metalloproteinase-9 and -2-regulated differentiation of HTB94 chondrosarcoma cells through the p38 kinase and JNK pathways.

doi: 10.3892/or.2014.3192

Figure Lengend Snippet: Figure 6. Effects of resveratrol on MMP-9-mediated regulation of type II collagen expression in HTB94 cells via the p38 kinase and JNK pathways. HTB94 cells were untreated (control) or treated with resveratrol in the absence or presence of 20 µM SB203580 or 20 µM SP600125 for 24 h. (A and B) Expression of MMP-9 and type Ⅱ collagen was detected by immunofluorescence staining. Statistically significant differences were noted between the control and the treatment groups. These data are the results of a typical experiment.

Article Snippet: Antibodies specific for MMP-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP-9 (Santa Cruz Biotechnology), type II collagen (Santa Cruz Biotechnology), SOX-9 (Santa Cruz Biotechnology), phosphorylated (p)p38 (Cell Signaling Technology, Danvers, MA, USA), p38 (Santa Cruz Biotechnology), pJNK (Cell Signaling Technology), JNK (Santa Cruz Biotechnology) and actin (Santa Cruz Biotechnology) were used for the experiments.

Techniques: Expressing, Control, Immunofluorescence, Staining

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 1 Higher ERK1/2 and p38 phosphorylation levels in dormant compared with aggressive T-ALL tumor samples inversely correlate with MKP-1 expression levels. (a) Detection of total and phosphorylated levels of ERK1/2, p38 and MKP-1 by western blot analysis of lysates from dormant or aggressive tumor samples formed by s.c. injection of MOLT-3 cells in NOD/SCID mice in the absence or the presence of exogenous bFGF (100 ng/ml), respectively.21 Membranes were probed with anti-a-tubulin as a loading control. Five representative samples per group are shown. (b) Columns report the mean values±s.d. of phosphorylated ERK (P-ERK)/ERK, P-p38/p38 (left), MKP-1/a-tubulin (right) ratios in all samples analyzed (n ¼ 10 per group). Samples were normalized to the ratio measured in the weakest one of the series, which was set at 1. Signal intensity was measured using a Bio-Rad XRS chemioluminescence detection system. *Po0.05 compared with dormant tumor values. (c) Expression of MKP-1 mRNA is similar in growing compared with dormant tumors. Total RNA was extracted from growing and dormant tumors (n ¼ 7 samples per group) and MKP-1 expression was determined by quantitative PCR. Columns represent mean values±s.d. MOLT-3 cells in vitro were used as reference sample to calculate the relative expression indicated in the panels. (d) MKP-1 is broadly expressed in T-ALL cell lines. Western blot analysis of MKP-1 in a panel of T-ALL cell lines; a-tubulin was used as loading control.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Phospho-proteomics, Expressing, Western Blot, Injection, Control, Real-time Polymerase Chain Reaction, In Vitro

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 2 Notch3 signalling regulates MKP-1 expression. (a) Treatment of T-ALL cell lines with GSIs lowers MKP-1 protein expression. MOLT-3, Jurkat, DND41 and CEM cells were treated for 72 h with CompE (10 mM) or solvent (dimethylsulphoxide (DMSO)) before western blot analysis. Columns represent mean values±s.d. of n ¼ 3 experiments. Cells cultivated in vitro in the presence of DMSO were used as reference sample to calculate the relative expression indicated in the panels. (b) Forced Notch3 ICD expression affects MKP-1 protein expression in MOLT-3 cells. Western blot analysis of MOLT-3 cells transfected by a Notch3 ICD expression plasmid (N3-ICD) versus control cells (enhanced green fluorescent protein (EGFP)). Density values were normalized to the ratio measured in the control sample, which was set at 1. (c) Notch3 silencing reduces MKP-1 expression and activates p38 in T-ALL cell lines. Left panel, western blot analysis of MOLT-3 cells transduced by lentiviral vectors encoding two different Notch3-specific shRNA (shN3/1 and shN3/2) or the control shRNA. In all samples analyzed, density values were normalized to the ratio measured in the control sample, which was set at 1. Mid panel: following shN3/1 transduction of DND41 cells, Notch3, MKP-1, p38, P-p38 and a-tubulin levels were analyzed by immunoblotting, and quantification was performed by P-p38 AlphaScreen SureFire kit (right panel), as described in section ‘Materials and methods’. Results are expressed as mean±s.d. of two independent experiments; *Po0.05.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Expressing, Solvent, Western Blot, In Vitro, Transfection, Plasmid Preparation, Control, shRNA, Transduction, Amplified Luminescent Proximity Homogenous Assay

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 4 MKP-1 modulates apoptosis of T-ALL cell lines. (a) Left panel: MKP-1 is expressed at higher levels in MOLT-3 primary cultures established from growing (GT) compared with dormant (DT) tumors by Western blot analysis. Right panel: Reduced apoptosis in primary cultures from GT compared with DT tumors following 6-h treatment with anysomicin (0.67 mg/ml). Apoptosis was evaluated by Annexin V staining and flow cytofluorimetric analysis. The columns report the mean values±s.d. of three independent experiments. *Po0.05 compared with untreated controls. (b) Transduction of MOLT-3 cells with lentiviral vectors encoding human MKP-1 (cytomegalovirus (CMV)–MKP-1) or shRNA targeting MKP-1 (shMKP-1) modulates MKP-1 expression levels in MOLT-3 cells, compared with controls (CMV; shRNA). The panels show the results of immunofluorescence analysis of MKP-1 expression in MOLT-3 cells transduced by the indicated constructs. Magnification, 200. (c) Detection of total and phosphorylated levels of JNK1-2 and p38 by Western blot analysis of lysates from MOLT-3 cells bearing modulated MKP-1 levels, treated or not with anisomycin. Membranes were probed with anti-b-actin as a loading control. One representative experiment of three performed is shown. (d) Measurements of apoptosis by Annexin V staining of T-ALL cells bearing increased (left panel) or reduced MKP-1 levels (right panels). MOLT-3 cells were treated for 6 h with anysomicin, 24 h with cisplatin (6 mg/ml), or 48-h serum starvation (fetal calf serum (FCS)) before staining with Annexin V–fluorescein isothiocyanate (FITC) and cytofluorimetric analysis. The columns report the mean values±s.d. of four independent experiments. *Po0.05 compared with controls. (e) Measurements of apoptosis of T-ALL cells bearing reduced MKP-1 levels following treatment with chemotherapeutics. Cell lines were treated for 24 h (MOLT-3) or 48 h (DND41) with cytarabine (20 nM), vincristine (10 mg/ml) or methotrexate (MTX, 300 nM) before staining with Annexin V–FITC and cytofluorimetric analysis. The columns report the mean values±s.d. of four independent experiments. *Po0.05 compared with controls.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Western Blot, Staining, Transduction, shRNA, Expressing, Construct, Control

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 5 MKP-1 attenuation modulates apoptosis and P-p38 levels in vivo. MOLT-3 or DND41 cells bearing normal or reduced MKP-1 levels were mixed with Matrigel and bFGF and injected s.c. into NOD/SCID mice (six animals per group). Before injection, cells were subjected to measurements of MKP-1 RNA levels, which indicated reduction of MKP-1 expression by 50% in shRNA targeting MKP-1 (shMKP-1)-transduced cells compared with shRNA controls (not shown). (a) Macroscopical analysis of s.c. MOLT-3 and DND41 tumors, 3-week after injection. (b) Measurements of apoptosis by Annexin V staining of MOLT-3 and DND41 tumors bearing normal or reduced MKP-1 levels. (c) Cytofluorimetric analysis of P-p38 levels in DND41 xenografts. The columns report the mean values±s.d. of 5–8 samples per group. *Po0.05 compared with controls.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: In Vivo, Injection, Expressing, shRNA, Staining

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 7 Expression of MKP-1 in primary T-ALL cells and correlation with the other molecular variables. (a) p38 phosphorylation in pediatric T-ALL samples inversely correlates with MKP-1 expression levels by western blot analysis. Membranes were probed with anti-b-actin as a loading control. (b) Notch 3 transcript levels by quantitative PCR in T-ALL samples characterized by low versus high MKP-1 levels. Results were normalized to the Notch3 transcript level in samples PDTALL6, which was set at 1. (c) Treatment of T-ALL cells with a g-secretase inhibitor (CompE) lowers MKP-1 protein expression. Primary cells were treated for 72 h with CompE (10 mM) or solvent (dimethylsulphoxide (DMSO)) before western blot analysis. Cells cultivated in vitro in the presence of DMSO were used as reference sample. Columns represent mean values±s.d. of the pooled experiments. *Po0.05.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Real-time Polymerase Chain Reaction, Solvent, In Vitro

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

doi: 10.1152/ajplung.00046.2012

Figure Lengend Snippet: Fig. 3. Inhibition of ERK1/2 and p38 MAPK affects CSE- and acrolein-induced IL-8 ex- pression in HBSMCs. A: cells pretreated with U0126 (10 M) or SB203580 (10 M) for 30 min were incubated with CSE (0.075 OD) or vehicle (Veh) for 8 h. Total RNA was extracted and relative IL-8 amount was measured by TaqMan real-time PCR. IL-8 mRNA levels are expressed as fold change over the basal after normalization to -actin. Each bar is the mean SE of 3 independent experiments. *Statistically different from basal, P 0.01. #Statistically different from CSE-treated cells, P 0.01. B–G: concen- tration-dependent inhibition by U0126, SB203580, and losmapimod of CSE (0.075 OD)- or acrolein (30 M)-evoked IL-8 re- lease in HBSMCs. Each point represents the mean SE of n 3 independent experi- ments performed in quadruplicate.

Article Snippet: Blots were then incubated with primary antibodies to vinculin (goat anti-vinculin, 1:333 dilution, Santa Cruz Biotechnology) and phospho-MK2 (rabbit anti-phosphoMK2, Thr222, 1:333 dilution, Cell Signaling Technology), p38 MAP kinase (rabbit anti p-38 MAPK, Cell Signaling Technology), and phospho-p38 (mouse anti-phospho-p38 MAPK, Thr180/Tyr182, Cell Signaling Technology), at room temperature for 20 min. With vacuum running continuously, blots were washed three times with TBS-T, prior to addition of the secondary antibodies IrDye 800CW donkey anti-goat or AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00046.2012 • www.ajplung.org IrDye 680CW goat anti-rabbit from LI-COR Biosciences (Lincoln, NE) for 10 min. Fluorescence signal was detected with an Odyssey scanner (LI-COR Biosciences).

Techniques: Inhibition, Incubation, Real-time Polymerase Chain Reaction

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

doi: 10.1152/ajplung.00046.2012

Figure Lengend Snippet: Fig. 4. Inhibition of MAPK-activated kinase 2 (MK2) affects CSE- and acrolein-induced IL-8 expression in HBSMCs. A–D: concen- tration-dependent inhibition by MK2 inhib- itor III and PF-3644022 of CSE (0.075 OD)- or acrolein (30 M)-evoked IL-8 release in HBSMCs. Each point represents the mean SE of n 3 independent experiments per- formed in quadruplicate.

Article Snippet: Blots were then incubated with primary antibodies to vinculin (goat anti-vinculin, 1:333 dilution, Santa Cruz Biotechnology) and phospho-MK2 (rabbit anti-phosphoMK2, Thr222, 1:333 dilution, Cell Signaling Technology), p38 MAP kinase (rabbit anti p-38 MAPK, Cell Signaling Technology), and phospho-p38 (mouse anti-phospho-p38 MAPK, Thr180/Tyr182, Cell Signaling Technology), at room temperature for 20 min. With vacuum running continuously, blots were washed three times with TBS-T, prior to addition of the secondary antibodies IrDye 800CW donkey anti-goat or AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00046.2012 • www.ajplung.org IrDye 680CW goat anti-rabbit from LI-COR Biosciences (Lincoln, NE) for 10 min. Fluorescence signal was detected with an Odyssey scanner (LI-COR Biosciences).

Techniques: Inhibition, Expressing

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

doi: 10.1152/ajplung.00046.2012

Figure Lengend Snippet: Fig. 5. CSE- and acrolein-evoked MK2 phosphorylation is regulated by p38 MAPK, but not by ERK1/2, signaling pathway in HBSMCs. Representative blots of MK2 immunoreactivity in HBSMCs treated with CSE (0.075 OD) or acrolein (30 M) and harvested at the indicated time points (A and B, respectively). Effects of a range of concentrations (0.1, 1, and 10 M) of the p38/ MAPK inhibitor SB203580 (C and D, respectively) and 10 M of the MEK1/2-ERK1/2 inhibitors U0126 and FR180204 (E and F, respectively) on CSE- and acrolein-induced MK2 phosphorylation. Quantitative densitometry values (fold change over basal of phospho-MK2/vinculin) are reported below each lane.

Article Snippet: Blots were then incubated with primary antibodies to vinculin (goat anti-vinculin, 1:333 dilution, Santa Cruz Biotechnology) and phospho-MK2 (rabbit anti-phosphoMK2, Thr222, 1:333 dilution, Cell Signaling Technology), p38 MAP kinase (rabbit anti p-38 MAPK, Cell Signaling Technology), and phospho-p38 (mouse anti-phospho-p38 MAPK, Thr180/Tyr182, Cell Signaling Technology), at room temperature for 20 min. With vacuum running continuously, blots were washed three times with TBS-T, prior to addition of the secondary antibodies IrDye 800CW donkey anti-goat or AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00046.2012 • www.ajplung.org IrDye 680CW goat anti-rabbit from LI-COR Biosciences (Lincoln, NE) for 10 min. Fluorescence signal was detected with an Odyssey scanner (LI-COR Biosciences).

Techniques: Phospho-proteomics

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

doi: 10.1152/ajplung.00046.2012

Figure Lengend Snippet: Fig. 6. CSE- and acrolein-evoked p38 MAPK and MK2 phosphorylation in small airway epithelial cells (SAECs) and normal human bronchial epithelial cells (NHBEs). Representative blots of p38 MAPK and MK2 immunoreactivity in SAECs (A–D) and NHBEs (E–H) treated with CSE (0.075 OD) or acrolein (30 M) and harvested at the indicated time points. Quantitative densitometry values (fold change over basal of phospho-p38/total-p38 or phospho-MK2/vinculin) are reported below each lane.

Article Snippet: Blots were then incubated with primary antibodies to vinculin (goat anti-vinculin, 1:333 dilution, Santa Cruz Biotechnology) and phospho-MK2 (rabbit anti-phosphoMK2, Thr222, 1:333 dilution, Cell Signaling Technology), p38 MAP kinase (rabbit anti p-38 MAPK, Cell Signaling Technology), and phospho-p38 (mouse anti-phospho-p38 MAPK, Thr180/Tyr182, Cell Signaling Technology), at room temperature for 20 min. With vacuum running continuously, blots were washed three times with TBS-T, prior to addition of the secondary antibodies IrDye 800CW donkey anti-goat or AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00046.2012 • www.ajplung.org IrDye 680CW goat anti-rabbit from LI-COR Biosciences (Lincoln, NE) for 10 min. Fluorescence signal was detected with an Odyssey scanner (LI-COR Biosciences).

Techniques: Phospho-proteomics

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

doi: 10.1152/ajplung.00046.2012

Figure Lengend Snippet: Fig. 7. CSE and acrolein act via p38 MAPK-mediated pathway to increase the stability of IL-8 mRNA in HBSMCs, SAECs, and alveolar macrophages (AMs) derived from a mixed population of smokers and COPD patients. HBSMCs, SAECs, and AMs were treated for 4 h with CSE (0.075 OD) (A–C, respectively) or acrolein (30 M) (D–F, respectively), followed by the addition of actinomycin D (5 g/ml) either alone or in presence of the p38/ MAPK inhibitor SB203580 (10 M). At the indicated times, total RNA was prepared and the remaining IL-8 mRNA was analyzed by TaqMan real-time PCR and normalized to -actin mRNA. Each point represents the mean SE of n 3 independent experiments. *Statistically different from basal (vehicle-treated), P 0.01.

Article Snippet: Blots were then incubated with primary antibodies to vinculin (goat anti-vinculin, 1:333 dilution, Santa Cruz Biotechnology) and phospho-MK2 (rabbit anti-phosphoMK2, Thr222, 1:333 dilution, Cell Signaling Technology), p38 MAP kinase (rabbit anti p-38 MAPK, Cell Signaling Technology), and phospho-p38 (mouse anti-phospho-p38 MAPK, Thr180/Tyr182, Cell Signaling Technology), at room temperature for 20 min. With vacuum running continuously, blots were washed three times with TBS-T, prior to addition of the secondary antibodies IrDye 800CW donkey anti-goat or AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00046.2012 • www.ajplung.org IrDye 680CW goat anti-rabbit from LI-COR Biosciences (Lincoln, NE) for 10 min. Fluorescence signal was detected with an Odyssey scanner (LI-COR Biosciences).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

doi: 10.1152/ajplung.00046.2012

Figure Lengend Snippet: Fig. 8. CSE acts via p38 MAPK/MK2, but not via ERK1/2, pathway to increase the stability of IL-8 mRNA in HBSMCs. HBSMCs were treated for 4 h with CSE (0.075 OD), followed by the addition of actinomycin D (5 g/ml), either alone, in presence of a MK2 inhibitor (MK2 inhibitor III or PF-3644022, 10 M) (A and B), or in presence of a MEK1/2-ERK1/2 inhibitor (U0126 or FR180204, 10 M) (C). At the indicated times, total RNA was prepared and the remaining IL-8 mRNA was analyzed by TaqMan real-time PCR and normalized to -actin mRNA. Each point represents the mean SE of n 3 independent experiments. *Statistically different from basal (vehicle- treated), P 0.01.

Article Snippet: Blots were then incubated with primary antibodies to vinculin (goat anti-vinculin, 1:333 dilution, Santa Cruz Biotechnology) and phospho-MK2 (rabbit anti-phosphoMK2, Thr222, 1:333 dilution, Cell Signaling Technology), p38 MAP kinase (rabbit anti p-38 MAPK, Cell Signaling Technology), and phospho-p38 (mouse anti-phospho-p38 MAPK, Thr180/Tyr182, Cell Signaling Technology), at room temperature for 20 min. With vacuum running continuously, blots were washed three times with TBS-T, prior to addition of the secondary antibodies IrDye 800CW donkey anti-goat or AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00046.2012 • www.ajplung.org IrDye 680CW goat anti-rabbit from LI-COR Biosciences (Lincoln, NE) for 10 min. Fluorescence signal was detected with an Odyssey scanner (LI-COR Biosciences).

Techniques: Real-time Polymerase Chain Reaction