Journal:

Article Title: Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression

doi:

Figure Lengend Snippet: Estrogen suppresses RANKL-induced activation of c-Jun and AP-1-mediated transcription in RAW264.7 cells. (A) Cells were treated for the indicated times with RANKL (80 ng/ml) or with the indicated concentrations of RANKL (RL) for 15 min and lysates examined for both JNK1 protein by Western blot and kinase activity (using GST-cJun as substrate). (B) Cells were treated for the indicated times with RANKL (80 ng/ml) and then subjected to Western blot analysis by using antibodies to c-Jun or phospho-c-Jun. Arrows indicate c-Jun and phospho-c-Jun forms. (C) Cells were transfected with a luciferase reporter plasmid (p36) containing three copies of an AP-1 response element. Cells were treated with vehicle or M-CSF and RANKL in the absence or presence of 17β-estradiol (10−8 M). Cells were harvested 24 h later and lysates assessed for luciferase and β-galactosidase activities and protein. Numbers represent the mean ± SE, n = 3 (a is significant vs. b and c; and b is significant vs. c at P ≤ 0.05). The results are representative of three independent experiments. (D) RAW264.7 cells were cotransfected with pFC2-luc and one of the following plasmids: pFA2-cJun (c-Jun), pFA-cFos (c-Fos), pFA-ATF2 (ATF2), or pFC-dbd (Gal4). Following transfection, cells were treated with RANKL in the presence of vehicle, 17β-estradiol (10−8 M), 4-hydroxytamoxifen (10−7 M), or raloxifene (10−7 M). Cells were harvested 24 h later and lysates assessed for luciferase activity, β-galactosidase activity, and protein. Numbers represent the mean ± SE, n = 3 (b is significant vs a and c at P ≤ 0.05). The results are representative of at least three independent experiments.

Article Snippet: Western blot analysis was used to assess the levels of JNK1 protein in total cell lysates using anti-JNK1 Abs (Santa Cruz Biotechnologies). c-Jun was detected in nuclear extracts by similar methods by using anti-c-Jun and anti-phospho-cJun Abs from New England Biolabs.

Techniques: Activation Assay, Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal:

Article Title: Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression

doi:

Figure Lengend Snippet: Estrogen suppresses JNK1 activity but does not alter RANK and c-Fms expression in RAW 264.7 cells. (A) Cells were pretreated with vehicle or 17β-estradiol (10−8 M) (15 min or 24 h) and then stimulated for 5 min with RANKL (80 ng/ml). Cell lysates were examined for JNK1 activity. RANKL-induced JNK1 activity was reduced 0 and 30% following a 15-min and 24-h treatment with estrogen, respectively. (B) Cells were treated for 24 h with vehicle, 17β-estradiol (10−8 M), 4-hydroxytamoxifen (10−7 M), or raloxifene (10−7 M) and then stimulated for 5 min with RANKL (80 ng/ml). Extracts were examined for JNK1 activity and by Western blot analysis. (C) RNA was isolated from cells treated for 24 h with either vehicle or 17β-estradiol (10−8 M) and subjected to RT-PCR by using oligonucleotide primer pairs for murine RANK, c-Fms, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The cycle-dependent appearance of specific DNA products was assessed by agarose gel electrophoresis. d, Cells were treated for 24 h with either vehicle or 17β-estradiol (10−8 M), and stimulated for 15 min with RANKL and nuclear extracts subjected to Western blot analysis by using Abs to c-Jun.

Article Snippet: Western blot analysis was used to assess the levels of JNK1 protein in total cell lysates using anti-JNK1 Abs (Santa Cruz Biotechnologies). c-Jun was detected in nuclear extracts by similar methods by using anti-c-Jun and anti-phospho-cJun Abs from New England Biolabs.

Techniques: Activity Assay, Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: British Journal of Pharmacology

Article Title: Cannabinoid CB 2 receptor attenuates morphine-induced inflammatory responses in activated microglial cells

doi: 10.1111/j.1476-5381.2012.01948.x

Figure Lengend Snippet: Morphine-enhanced Akt and ERK1/2 phosphorylation in a µ-opioid receptor-dependent manner. (A), Western blot analysis of Akt, ERK1/2, p38, and JNK1/2 phosphorylation in primary microglial cells incubated for 30 min with morphine (MOR) (1–1000 nM). The immunoblot signals were quantified using a VersaDoc Imaging System (Bio-Rad). The ratio of phospho-protein to total protein is used. The mean values of four independent experiments (one of which is shown) were normalized to the result obtained with morphine-untreated cell cultures (0). Densitometric analysis is shown. The unstimulated control (0, cells in the absence of morphine) was set to 100%. Data shown are mean ± SE values of four separate experiments performed in triplicate (n= 4). *P < 0.05 significantly different from unstimulated control; analysis was by anova followed by Dunnett's test. (B), Primary microglial cells were treated with morphine (100 nM) for 0, 15, 30 and 60 min, subjected to Western blot analysis and probed with anti-pERK1/2, anti-pAkt, then Akt and ERK1/2 antibody. The ratio of phospho-protein to total protein is used. Data shown are mean ± SE values of four separate experiments performed in triplicate (n= 4). *P < 0.01 significantly different from control conditions (0); #P < 0.01 significantly different from LPS conditions; analysis was by anova followed by Dunnett's test. (C), Image of Western blot membrane probed with anti-pERK1/2, anti-pAkt, then Akt and ERK1/2 antibody of primary microglial cells pre-treated for 30 min with 0 or 100 nM of the µ-opioid receptor antagonist CTAP, then treated for 30 min with 0 or 100 nM morphine. The ratio of phospho-protein to total protein is used. Data shown are mean ± SEM values of four separate experiments performed in triplicate (n= 4). *P < 0.01 significantly different from untreated cells; analysis was by anova followed by Dunnett's test.

Article Snippet: Phosphorylated (Thr 180 /Tyr 182 ) and total p38, phosphorylated (Thr 183 /Tyr 185 ) and total JNK1/2 antibodies were from Cell Signalling Technology (Celbio, Milan, Italy).

Techniques: Western Blot, Incubation, Imaging

Journal: British Journal of Pharmacology

Article Title: Cannabinoid CB 2 receptor attenuates morphine-induced inflammatory responses in activated microglial cells

doi: 10.1111/j.1476-5381.2012.01948.x

Figure Lengend Snippet: Effect of CB2 receptor stimulation in morphine-treated activated primary microglial cells. JWH-015 and morphine (MOR) effect on Akt, ERK1/2, p38 and JNK1/2 phosphorylation in primary microglial cells treated with LPS. Microglial cells were incubated with DMSO vehicle (CTR), with MOR (100 nM), or with JWH-015 (100 nM) alone and in combination in the presence of LPS 1 µg·mL−1 for 30 min. The mean values of four independent experiments (one of which is shown) were normalized to the result obtained in cells in the absence of LPS (CTR). Data shown are mean ± SEM values of four separate experiments performed in triplicate (n= 4). CTR was set to 100%. The immunoblot signals were quantified using a VersaDoc Imaging System (Bio-Rad). Densitometric analysis of kinase activation is shown. The ratio of phospho-protein to total protein is used. *P < 0.05 significantly different from CTR; #P < 0.05 significantly different from cells treated with LPS; §P < 0.05 significantly different from cells treated with LPS + morphine; analysis was by anova followed by Dunnett's test.

Article Snippet: Phosphorylated (Thr 180 /Tyr 182 ) and total p38, phosphorylated (Thr 183 /Tyr 185 ) and total JNK1/2 antibodies were from Cell Signalling Technology (Celbio, Milan, Italy).

Techniques: Incubation, Western Blot, Imaging, Activation Assay

Journal:

Article Title: A Protein Kinase C?-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

doi: 10.2353/ajpath.2009.080180

Figure Lengend Snippet: Phosphorylation of ERK1/2 and JNK1/2 is decreased after silencing of PKD in C10 lung epithelial cells. C10 cells were transfected with siPKD or scramble control construct before addition of asbestos for 10 hours. Cells were homogenized and Western blot analyses were performed using antibodies to p-ERK1/2 and total ERK1/2 (A and B); p-JNK1/2 and total JNK1/2 (C and D). A and C: Representative Western blots. B and D: Quantitation of data, as described in Materials and Methods. Results are typical of three independent experiments.*P ≤ 0.05 in comparison with sham group (0). †P ≤ 0.05 in comparison with siControl group. Bars = Mean ± SEM.

Article Snippet: Western blots were performed as described previously, 7 using antibodies specific to total and phosphorylated PKD (rabbit polyclonal anti-PKD, 1:1000, rabbit polyclonal anti-phosphor-Ser 744/748 PKD 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated ERK1/2 (rabbit polyclonal anti-ERK1/2, 1:1000; rabbit polyclonal phosphor-ERK1/2 1:500, and mouse monoclonal anti-ERKp42 1:1000; Cell Signaling Technology, Danvers, MA), total and phosphorylated JNK1/2 (rabbit polyclonal JNK1/2, 1:200, rabbit polyclonal phospho JNK1/2, 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated c-Jun (rabbit polyclonal c-Jun at 1:1000, rabbit polyclonal phospho c-Jun at 1:500, Cell Signaling Technology, Danvers, MA), total PKCδ (rabbit polyclonal PKCδ at 1:1000, Santa Cruz, Santa Cruz, CA), total β-Actin 1:2000 (Abcam, Cambridge, MA), and total Bim (rabbit polyclonal anti-Bim at 1:500, Cell Signaling Technology, Danvers, MA).

Techniques: Transfection, Construct, Western Blot, Quantitation Assay

Journal:

Article Title: A Protein Kinase C?-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

doi: 10.2353/ajpath.2009.080180

Figure Lengend Snippet: BimEL (23kD) and BimL (16kD) proteins are decreased after addition of asbestos to C10 lung epithelial cells (A, B) and up-regulated after silencing of PKD (C) or inhibition of ERK1/2 using U0126 (D). A: Representative Western blot and (B) quantitation of data. C: Quantitative data showing that BimEL (23kD) is increased in siPKD transfected cells. D: Quantitative data showing that U0126, but not SP600125, prevents asbestos-associated decreases in BimEL. The ERK1/2 inhibitor, U0126 (10 μmol/L), and JNK1/2 inhibitor, SP600125 (SP; 10 μmol/L) were added before asbestos as described in Materials and Methods. *P ≤ 0.05 in comparison with unexposed cells (0); †P ≤ 0.05 in comparison with siControl at same time point. Bars = Mean ± SEM.

Article Snippet: Western blots were performed as described previously, 7 using antibodies specific to total and phosphorylated PKD (rabbit polyclonal anti-PKD, 1:1000, rabbit polyclonal anti-phosphor-Ser 744/748 PKD 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated ERK1/2 (rabbit polyclonal anti-ERK1/2, 1:1000; rabbit polyclonal phosphor-ERK1/2 1:500, and mouse monoclonal anti-ERKp42 1:1000; Cell Signaling Technology, Danvers, MA), total and phosphorylated JNK1/2 (rabbit polyclonal JNK1/2, 1:200, rabbit polyclonal phospho JNK1/2, 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated c-Jun (rabbit polyclonal c-Jun at 1:1000, rabbit polyclonal phospho c-Jun at 1:500, Cell Signaling Technology, Danvers, MA), total PKCδ (rabbit polyclonal PKCδ at 1:1000, Santa Cruz, Santa Cruz, CA), total β-Actin 1:2000 (Abcam, Cambridge, MA), and total Bim (rabbit polyclonal anti-Bim at 1:500, Cell Signaling Technology, Danvers, MA).

Techniques: Inhibition, Western Blot, Quantitation Assay, Transfection

Journal:

Article Title: A Protein Kinase C?-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

doi: 10.2353/ajpath.2009.080180

Figure Lengend Snippet: Immunoprecipitation (IP) experiments showing association of PKCδ, PKD, and ERK1/2 (A, B) and PKD, JNK1/2, and ERK1/2 (C, D) after immunoprecipitation of protein using antibodies to PKD (A, B) or JNK1/2 (C, D) respectively, in C10 lung epithelial cells. A and C show representative Western blots; and B and D show quantitation of data from duplicate experiments. *P ≤ 0.05 in comparison with respective groups at 0 time. IgG was used as a loading control in C and D. IB = Immunoblotting. Bars = Mean ± SEM.

Article Snippet: Western blots were performed as described previously, 7 using antibodies specific to total and phosphorylated PKD (rabbit polyclonal anti-PKD, 1:1000, rabbit polyclonal anti-phosphor-Ser 744/748 PKD 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated ERK1/2 (rabbit polyclonal anti-ERK1/2, 1:1000; rabbit polyclonal phosphor-ERK1/2 1:500, and mouse monoclonal anti-ERKp42 1:1000; Cell Signaling Technology, Danvers, MA), total and phosphorylated JNK1/2 (rabbit polyclonal JNK1/2, 1:200, rabbit polyclonal phospho JNK1/2, 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated c-Jun (rabbit polyclonal c-Jun at 1:1000, rabbit polyclonal phospho c-Jun at 1:500, Cell Signaling Technology, Danvers, MA), total PKCδ (rabbit polyclonal PKCδ at 1:1000, Santa Cruz, Santa Cruz, CA), total β-Actin 1:2000 (Abcam, Cambridge, MA), and total Bim (rabbit polyclonal anti-Bim at 1:500, Cell Signaling Technology, Danvers, MA).

Techniques: Immunoprecipitation, Western Blot, Quantitation Assay

Journal:

Article Title: A Protein Kinase C?-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

doi: 10.2353/ajpath.2009.080180

Figure Lengend Snippet: Immunofluorescence experiments showing simultaneous induction of H2O2-induced pJNK1/2 and pERK1/2 signaling in C10 lung epithelial cells. (A) shows untreated confluent C10 cells. B and C show C10 cells exposed to 0.5 mmol/L and 5 mmol/L H2O2 for 30 minutes. (D) illustrates the lack of p-JNK1/2 or p-ERK1/2 staining in cells stained with secondary antibody alone. Nuclei are stained with Sytox Green. Red dots indicate p-ERK1/2 phosphorylation. All micrographs at original magnification ×400. Bar = 50 mmol/L.

Article Snippet: Western blots were performed as described previously, 7 using antibodies specific to total and phosphorylated PKD (rabbit polyclonal anti-PKD, 1:1000, rabbit polyclonal anti-phosphor-Ser 744/748 PKD 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated ERK1/2 (rabbit polyclonal anti-ERK1/2, 1:1000; rabbit polyclonal phosphor-ERK1/2 1:500, and mouse monoclonal anti-ERKp42 1:1000; Cell Signaling Technology, Danvers, MA), total and phosphorylated JNK1/2 (rabbit polyclonal JNK1/2, 1:200, rabbit polyclonal phospho JNK1/2, 1:1000, Cell Signaling Technology, Danvers, MA), total and phosphorylated c-Jun (rabbit polyclonal c-Jun at 1:1000, rabbit polyclonal phospho c-Jun at 1:500, Cell Signaling Technology, Danvers, MA), total PKCδ (rabbit polyclonal PKCδ at 1:1000, Santa Cruz, Santa Cruz, CA), total β-Actin 1:2000 (Abcam, Cambridge, MA), and total Bim (rabbit polyclonal anti-Bim at 1:500, Cell Signaling Technology, Danvers, MA).

Techniques: Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Activin B Stimulates Mouse Vibrissae Growth and Regulates Cell Proliferation and Cell Cycle Progression of Hair Matrix Cells through ERK Signaling

doi: 10.3390/ijms20040853

Figure Lengend Snippet: Activin B stimulated JNK, ERK, and P38 phosphorylation in hair matrix cells. ( A , B ) The plot and the relative quantification of the expression of phosphorylation of ERK in HHGMCs treated with 10 ng/mL Activin B for 15 min, 30 min, 2 h, and 4 h. ( C , D ) The plot and the relative quantification of the expression of phosphorylation of JNK in cells treated with 10 ng/mL Activin B for different times. ( E , F ) The plot and the relative quantification of the expression of phosphorylation of P38 in cells treated with 10 ng/mL Activin B for different times. * p < 0.05; ** p < 0.01, compared with the PBS group. Three independent experiments were conducted per data point. All error bars indicate SEM.

Article Snippet: The blots were then washed with stripping buffer (Millipore, Bedford, MA, USA) for 10 min at room temperature and re-probed with antibodies against total-JNK (1:1000; Cell Signaling, Danvers, MA, USA; #9258), total-ERK (1:1000; Cell Signaling, Danvers, MA, USA; #4695S), total-P38 (1:1000; Cell Signaling, Danvers, MA, USA; #9212S), or total-Elk1 (1:1000; Santa Cruz, Dallas, TX, USA; sc-65986).

Techniques: Phospho-proteomics, Quantitative Proteomics, Expressing