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  • 99
    Millipore monoclonal anti map kinase activated diphosphorylated erk 1 2 antibody
    Monoclonal Anti Map Kinase Activated Diphosphorylated Erk 1 2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 714 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc perk1 2
    Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3813 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti perk1 2
    KRAS Regulation of MYC Expression is Dependent on MEK-ERK1/2 Signaling, but Not PI3K-AKT-GSK3β (A) PDAC cell lines were transfected with NS or KRAS siRNA (24 hr). Immunoblotting of cell lysates was done to determine total KRAS and MYC, phosphorylated ERK1/2 (pERK) and its substrate p90RSK (pRSK), and phosphorylated AKT (pAKT) and its substrate GSK3β (pGSK3β). (B) PDAC cell lines were treated with the ERK1/2 inhibitor SCH772984 (ERKi) or the MEK1/2 inhibitor selumetinib (MEKi) for 24 hr and cell lysates were immunoblotted as indicated. (C) PDAC cell lines were treated with the PI3K inhibitor AZD8186 (PI3Ki) or the AKT inhibitor AZD5363 (AKTi) for 24 hr and cell lysates were immunoblotted as indicated. (D) IHC staining of PDAC patient samples was performed for total MYC, <t>pERK1/2</t> and pAKT. Representative sections are shown. Scale bar = 100 μm. (E) Kaplan-Meier survival curves for patients with high (IHC scores of 2+ or higher) or low (1+ or lower) levels of total MYC (left panel) or phosphorylated ERK1/2 (right panel). N=72 patients per group. .
    Anti Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology perk1 2
    Double immunofluorescent staining showing the co‐localization of <t>pERK1/2</t> and POMC proteins in the ARC of amphetamine‐treated rats. Frozen sections of the brain were plated on slides and stained with anti‐pERK1/2 and anti‐POCM antibodies in the hypothalamus. Under the analysis of confocal microscopy, the co‐localization image was seen by the merge of pERK1/2 (green) and POMC (red) immunofluorescent images, giving a yellow colour in the merged image panel. Blue: nucleus. Scale bar: 20 μm. 3rd V: the third ventricle.
    Perk1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 732 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti perk1 2
    Suppression of MAP kinase signalling in miR-21 morphants and BDM-treated hearts. ( a – c ) In the WT beating hearts, a phosphorylated form of Erk1/2 <t>(pErk1/2)</t> was stained in the cuboidal endocardial cells at the AV junction (yellow arrowheads in a ). The entire endocardial cells were labelled by GFP, which expression was driven by the fli1 promoter ( b , c ). ( d – f ) In the miR-21 morphants, the pErk1/2 staining was week and the cells were flat. ( g – i ) When the heartbeat was stopped by BDM, phosphorylation of Erk1/2 was also suppressed, leaving only faint staining in the apical surface of flattened endocardial cells. ( j – o ) Injection of the miR-21 target protection MO and the U0126 treatment gave the same results on pErk1/s staining. A, atrium; V, ventricle. Scale bars, 20 μm.
    Rabbit Anti Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 560 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti perk1 2
    CRH-elicited cAMP response depends on bicarbonate. (A) HT22-CRHR1 cells were stimulated with CRH in HCO 3 − -free or 25 mM HCO 3 − DMEM. (B) HT22-CRHR1 cells preincubated with vehicle (control) or anion channel blocker DIDS were stimulated with CRH. <t>pERK1/2</t> and total ERK1/2 were measured and expressed as the percentage of maximum pERK1/2 under control conditions (mean ± SEM, n = 3). *, P
    Anti Perk1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti perk1 2 antibody
    Glutamate injections upregulated pNR2B and <t>pERK1/2</t> expression in the hippocampus. OVX rats were assigned to 5 groups for western blot analyses. At 5, 15, 30, and 45 min following the glutamate injections, rats were sacrificed and the intact hippocampus was dissected. In addition, in control group, we eliminated the possibility that the needle insertion produced protein expression changes by sacrificing rats immediately after vehicle injections. (A,B) Representative western blot of pNR2B and pERK1/2 after glutamate injections. Quantification of protein levels was normalized against loading control β-actin and presented as the relative density compared with the control group. * P
    Anti Perk1 2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc perk1 2 t202 y204
    Effects of LY3009120 on signaling and gene expression ( A ) CRC cell lines were treated with DMSO, 0.1 μM or 1 μM LY3009120 for 30 minutes ( left panel ) or 2 hrs ( right panel ). Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. ( B ) CRC cell lines were treated with increasing concentrations of LY3009120 and fixed at the times indicated. Cells were stained with Hoechst stain and nuclei counts were obtained. At the 72 hr time point, replicate plates were assessed for proliferation by CTG analysis. ( C ) CRC cell lines were treated with DMSO or logarithmic concentrations of LY3009120 and fixed at 24 hrs post-treatment. Cells were stained for immunofluorescence analysis with Hoechst stain and antibodies against the proteins indicated and the average intensity of the signal for each analyte was measured by HCI. Results are plotted as percent of DMSO-treated cells and are representative of two independent experiments. The signal for <t>pERK1/2</t> <t>T202/Y204</t> and pS6 S240/244 was normalized to the levels of ERK1/2 and S6 respectively. ( D ) CRC cell lines were treated with DMSO or 1 μM LY3009120 for 24 and 48 hrs. Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. ( E ) CRC cell lines were treated with DMSO or increasing concentrations of LY3009120 for 24 hrs. Expression levels of the indicated genes were assessed by Affymetrix analysis.
    Perk1 2 T202 Y204, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam perk1 2
    L-NAME inhibits NR1 and ERK1/2 phosphorylation, as well as nNOS and iNOS upregulation in db/db mice. A: Representative immunoblots of pNR1, total NR1, and actin from the LSC of db+ and db/db mice intrathecally treated with control aCSF (C) or aCSF with L-NAME. L-NAME treatment decreased NR1 phosphorylation in db/db, but not db+ mice. B: Densitometric analysis of pNR1 immunoblots. L-NAME treatment decreased the levels of NR1 phosphorylation back to that of control db+ mice. C: Representative immunoblots of <t>pERK1/2,</t> ERK1/2, and actin from the LSC of db+ and db/db mice treated with control solution (C) or L-NAME. L-NAME treatment decreased ERK1/2 phosphorylation in db/db, but not db+ mice. D: Densitometric analysis of pERK1/2 immunoblots. L-NAME treatment decreased the levels of ERK1/2 phosphorylation back to control levels. E: nNOS immunoblots revealed that L-NAME treatment reduced nNOS upregulation in db/db mice. F: Densitometric analysis of nNOS immunoblots demonstrated L-NAME treatment decreased nNOS levels in db/db mice back to control levels. G: iNOS immunoblots showed that L-NAME treatment reduced iNOS upregulation in db/db mice. H: Densitometric analysis of iNOS immunoblots demonstrated iNOS treatment decreased iNOS levels in db/db mice back to control levels. **, p
    Perk1 2, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson perk1 2
    Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 <t>(pErk1/2)</t> positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p
    Perk1 2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p44 42 mapk perk1 2
    Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 <t>(pErk1/2)</t> positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p
    Phospho P44 42 Mapk Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse anti perk1 2
    Acute restraint stress prevents formalin-induced increases in pERK2 in the CeA of male mice Following restraint (or control habituation), male mice received an injection of either 2% formalin (n=6) or sterile saline (n=6) in the rear right paw. Three hours following injection, animals were sacrificed and CeA tissue was isolated. Using Western blotting techniques, ( A. ) the CeA was assessed for expression of <t>pERK1/2,</t> well characterized pain signaling molecules, and total ERK1/2. B. Formalin treatment caused a significant increase in pERK1 (Two-way ANOVA: effect of formalin, p
    Mouse Anti Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc perk1 2 thr202 tyr204
    Acute restraint stress prevents formalin-induced increases in pERK2 in the CeA of male mice Following restraint (or control habituation), male mice received an injection of either 2% formalin (n=6) or sterile saline (n=6) in the rear right paw. Three hours following injection, animals were sacrificed and CeA tissue was isolated. Using Western blotting techniques, ( A. ) the CeA was assessed for expression of <t>pERK1/2,</t> well characterized pain signaling molecules, and total ERK1/2. B. Formalin treatment caused a significant increase in pERK1 (Two-way ANOVA: effect of formalin, p
    Perk1 2 Thr202 Tyr204, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti perk1 2 thr202 tyr204
    Dose-response effect of Ang-(1–7) on ERK1/2 phosphorylation levels. HEK293T cells were transfected either with the pcDNA3.1/myc-MasR vector or with the empty vector (pcDNA 3.1), starved in DMEM without serum for 8 h and then incubated for 5 min with increasing concentrations of Ang-(1–7). (A) Phosphorylation levels of ERK1/2 at activating residues <t>Thr202/Tyr204,</t> total ERK1/2 and β-tubulin abundance were determined by Western Blotting. (B) Data quantification. Bands intensity was quantified by optical densitometry, each individual value of pERK was normalized to that of total ERK and expressed as relative to the value obtained after incubation with cells transfected with pcDNA 3.1 and incubated with vehicle (basal value). Data are means ± SEM of three independent experiments. Values were fitted to a sigmoidal dose-response curve. Maximal response best-fitted values were analyzed by extra sum-of-squares F test, significantly different ( P
    Anti Perk1 2 Thr202 Tyr204, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti perk1 2
    Dose-response effect of Ang-(1–7) on ERK1/2 phosphorylation levels. HEK293T cells were transfected either with the pcDNA3.1/myc-MasR vector or with the empty vector (pcDNA 3.1), starved in DMEM without serum for 8 h and then incubated for 5 min with increasing concentrations of Ang-(1–7). (A) Phosphorylation levels of ERK1/2 at activating residues <t>Thr202/Tyr204,</t> total ERK1/2 and β-tubulin abundance were determined by Western Blotting. (B) Data quantification. Bands intensity was quantified by optical densitometry, each individual value of pERK was normalized to that of total ERK and expressed as relative to the value obtained after incubation with cells transfected with pcDNA 3.1 and incubated with vehicle (basal value). Data are means ± SEM of three independent experiments. Values were fitted to a sigmoidal dose-response curve. Maximal response best-fitted values were analyzed by extra sum-of-squares F test, significantly different ( P
    Mouse Anti Perk1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse monoclonal antibody anti perk1 2
    Effect of zileuton and montelukast on ERK1/2 activation. Western blot analysis of extracts of spinal cord tissue collected at 24 h after injury. SCI caused an increase of the ERK1/2 phosphorylation in vehicle-treated mice. The treatment with zileuton or montelukast reduced <t>pERK1/2</t> levels. Densitometric analysis of protein expression represents the mean±s.e.mean of 10 spinal cord tissues. Data were normalized on the basis of ERK-2 levels. ° P
    Mouse Monoclonal Antibody Anti Perk1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega perk1 2
    Effect of treatment with the raf inhibitor SB386023-b in cerebral arteries on the protein levels of <t>pERK1/2</t> after SAH . There are increased expressions of the pERK1/2 protein levels in the SAH compared to the sham operated rats. Treatment with SB386023-b after 6 h prevented the increased protein expression. Data are presented as the pERK1/2/β-actin mean optical density ratio relative to control. Data are expressed as mean ± s.e.m. * P ≤ 0.05.
    Perk1 2, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    KRAS Regulation of MYC Expression is Dependent on MEK-ERK1/2 Signaling, but Not PI3K-AKT-GSK3β (A) PDAC cell lines were transfected with NS or KRAS siRNA (24 hr). Immunoblotting of cell lysates was done to determine total KRAS and MYC, phosphorylated ERK1/2 (pERK) and its substrate p90RSK (pRSK), and phosphorylated AKT (pAKT) and its substrate GSK3β (pGSK3β). (B) PDAC cell lines were treated with the ERK1/2 inhibitor SCH772984 (ERKi) or the MEK1/2 inhibitor selumetinib (MEKi) for 24 hr and cell lysates were immunoblotted as indicated. (C) PDAC cell lines were treated with the PI3K inhibitor AZD8186 (PI3Ki) or the AKT inhibitor AZD5363 (AKTi) for 24 hr and cell lysates were immunoblotted as indicated. (D) IHC staining of PDAC patient samples was performed for total MYC, pERK1/2 and pAKT. Representative sections are shown. Scale bar = 100 μm. (E) Kaplan-Meier survival curves for patients with high (IHC scores of 2+ or higher) or low (1+ or lower) levels of total MYC (left panel) or phosphorylated ERK1/2 (right panel). N=72 patients per group. .

    Journal: Cancer cell

    Article Title: KRAS Suppression-Induced Degradation of MYC is Antagonized by a MEK5-ERK5 Compensatory Mechanism

    doi: 10.1016/j.ccell.2018.10.001

    Figure Lengend Snippet: KRAS Regulation of MYC Expression is Dependent on MEK-ERK1/2 Signaling, but Not PI3K-AKT-GSK3β (A) PDAC cell lines were transfected with NS or KRAS siRNA (24 hr). Immunoblotting of cell lysates was done to determine total KRAS and MYC, phosphorylated ERK1/2 (pERK) and its substrate p90RSK (pRSK), and phosphorylated AKT (pAKT) and its substrate GSK3β (pGSK3β). (B) PDAC cell lines were treated with the ERK1/2 inhibitor SCH772984 (ERKi) or the MEK1/2 inhibitor selumetinib (MEKi) for 24 hr and cell lysates were immunoblotted as indicated. (C) PDAC cell lines were treated with the PI3K inhibitor AZD8186 (PI3Ki) or the AKT inhibitor AZD5363 (AKTi) for 24 hr and cell lysates were immunoblotted as indicated. (D) IHC staining of PDAC patient samples was performed for total MYC, pERK1/2 and pAKT. Representative sections are shown. Scale bar = 100 μm. (E) Kaplan-Meier survival curves for patients with high (IHC scores of 2+ or higher) or low (1+ or lower) levels of total MYC (left panel) or phosphorylated ERK1/2 (right panel). N=72 patients per group. .

    Article Snippet: The slides were incubated with the primary antibodies using the following conditions: anti-Myc (Abcam, Cat# ab32072, 1:75 for 60 min); anti-pERK1/2 (Cell Signaling Technology, Cat# 4376, 1:175 for 60 min); and anti-pan-AKT (phospho T308) (Abcam, Cat# ab38449, 1:50 for 60 min) with subsequent signal detection by the Mach 3 Rabbit HRP Polymer Detection kit (Biocare Medical).

    Techniques: Expressing, Transfection, Immunohistochemistry, Staining

    Double immunofluorescent staining showing the co‐localization of pERK1/2 and POMC proteins in the ARC of amphetamine‐treated rats. Frozen sections of the brain were plated on slides and stained with anti‐pERK1/2 and anti‐POCM antibodies in the hypothalamus. Under the analysis of confocal microscopy, the co‐localization image was seen by the merge of pERK1/2 (green) and POMC (red) immunofluorescent images, giving a yellow colour in the merged image panel. Blue: nucleus. Scale bar: 20 μm. 3rd V: the third ventricle.

    Journal: British Journal of Pharmacology

    Article Title: Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats) Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats

    doi: 10.1111/bph.14120

    Figure Lengend Snippet: Double immunofluorescent staining showing the co‐localization of pERK1/2 and POMC proteins in the ARC of amphetamine‐treated rats. Frozen sections of the brain were plated on slides and stained with anti‐pERK1/2 and anti‐POCM antibodies in the hypothalamus. Under the analysis of confocal microscopy, the co‐localization image was seen by the merge of pERK1/2 (green) and POMC (red) immunofluorescent images, giving a yellow colour in the merged image panel. Blue: nucleus. Scale bar: 20 μm. 3rd V: the third ventricle.

    Article Snippet: Antibodies against NPY, tERK1/2, pERK1/2 and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Staining, Confocal Microscopy

    Immunofluorescent staining of pERK1/2 in hypothalamic ARC and PVN regions in rats receiving saline or amphetamine (AMPH; 4 mg·kg −1 ; i.p.) treatment. Frontal sections through the median eminence level were obtained. Upper panel: photo images of (A–C) show sections in control group, photo images of (D–F) show pERK fluorescence in AMPH‐treated group and photo images of (G–I) show pERK (red) and nucleus (blue) fluorescence of double staining in AMPH‐treated group. Scale bars in images of (A, D and G) are 100 μm (100×), whereas those in (B, C, E, F, H and I) are 20 μm (400×). Lower panel: results of relative densitometric values ( n = 5 for each group; total of three groups) for pERK fluorescence intensity in PVN and ARC regions compared to the control group in AMPH‐treated rat. Each value represents mean ± SEM. * P

    Journal: British Journal of Pharmacology

    Article Title: Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats) Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats

    doi: 10.1111/bph.14120

    Figure Lengend Snippet: Immunofluorescent staining of pERK1/2 in hypothalamic ARC and PVN regions in rats receiving saline or amphetamine (AMPH; 4 mg·kg −1 ; i.p.) treatment. Frontal sections through the median eminence level were obtained. Upper panel: photo images of (A–C) show sections in control group, photo images of (D–F) show pERK fluorescence in AMPH‐treated group and photo images of (G–I) show pERK (red) and nucleus (blue) fluorescence of double staining in AMPH‐treated group. Scale bars in images of (A, D and G) are 100 μm (100×), whereas those in (B, C, E, F, H and I) are 20 μm (400×). Lower panel: results of relative densitometric values ( n = 5 for each group; total of three groups) for pERK fluorescence intensity in PVN and ARC regions compared to the control group in AMPH‐treated rat. Each value represents mean ± SEM. * P

    Article Snippet: Antibodies against NPY, tERK1/2, pERK1/2 and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Staining, Fluorescence, Double Staining

    Schematic illustration showing the flow in the suppressive action of amphetamine (AMPH). Amphetamine treatment activates dopamine release in the brain, which may in turn activate hypothalamic POMC expression, inhibit hypothalamic NPY expression and finally decrease food intake. The activation of POMC neurons, which is partly due to the disinhibition of NPY neurons, may result in the increased expression of pERK1/2, pCREB, POMC and MC 3 receptor (MC3R). Finally, the decrease in NPY or the increase in MC 3 receptors may contribute to the reduction of food intake. The thinner arrows indicate the flow of amphetamine treatment in the induction of anorexia. The solid black arrow indicates the activation, the dotted arrows indicate the inhibition and the dashed arrow represents the disinhibition (or activation). The upward and downward arrows in the squares indicate the responses of increase and decrease.

    Journal: British Journal of Pharmacology

    Article Title: Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats) Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats

    doi: 10.1111/bph.14120

    Figure Lengend Snippet: Schematic illustration showing the flow in the suppressive action of amphetamine (AMPH). Amphetamine treatment activates dopamine release in the brain, which may in turn activate hypothalamic POMC expression, inhibit hypothalamic NPY expression and finally decrease food intake. The activation of POMC neurons, which is partly due to the disinhibition of NPY neurons, may result in the increased expression of pERK1/2, pCREB, POMC and MC 3 receptor (MC3R). Finally, the decrease in NPY or the increase in MC 3 receptors may contribute to the reduction of food intake. The thinner arrows indicate the flow of amphetamine treatment in the induction of anorexia. The solid black arrow indicates the activation, the dotted arrows indicate the inhibition and the dashed arrow represents the disinhibition (or activation). The upward and downward arrows in the squares indicate the responses of increase and decrease.

    Article Snippet: Antibodies against NPY, tERK1/2, pERK1/2 and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Flow Cytometry, Expressing, Activation Assay, Inhibition

    The effect of daily amphetamine (2 mg·kg −1 ; i.p.) on the ratios of (A) hypothalamic pERK/tERK and pCREB/tCREB expression (upper panel), and on (B) NPY, POMC, pERK1/2, pCREB and MC 3 receptor (MC3R) (lower panel) expression over a 4 day period. Inserted images showed the results of Western blots, and the columns represent their relative densitometric values. Contents of protein in AMPH‐treated groups were indicated as the percentage of the control group. Bars are the means ± SEM; n = 8 for each daily treatment group (from Day 0 to Day 4; total of five day‐dependent groups). * P

    Journal: British Journal of Pharmacology

    Article Title: Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats) Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats

    doi: 10.1111/bph.14120

    Figure Lengend Snippet: The effect of daily amphetamine (2 mg·kg −1 ; i.p.) on the ratios of (A) hypothalamic pERK/tERK and pCREB/tCREB expression (upper panel), and on (B) NPY, POMC, pERK1/2, pCREB and MC 3 receptor (MC3R) (lower panel) expression over a 4 day period. Inserted images showed the results of Western blots, and the columns represent their relative densitometric values. Contents of protein in AMPH‐treated groups were indicated as the percentage of the control group. Bars are the means ± SEM; n = 8 for each daily treatment group (from Day 0 to Day 4; total of five day‐dependent groups). * P

    Article Snippet: Antibodies against NPY, tERK1/2, pERK1/2 and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot

    The effect of ERK antisense/amphetamine (AMPH) co‐administration on the changes in hypothalamic pERK1/2, pCREB, NPY, POMC and MC 3 receptor (MC3R) expression during a 4 day period of drug treatment. Upper panel: each of the protein levels was measured by Western blot. Lower panel: the protein levels in drug‐treated groups were indicated as the percentage of the control group (Day 0). Each densitometric value represents the mean ± SEM; n = 6 for each group (total of seven drug‐treated groups). * P

    Journal: British Journal of Pharmacology

    Article Title: Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats) Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide‐mediated appetite control in amphetamine‐treated rats

    doi: 10.1111/bph.14120

    Figure Lengend Snippet: The effect of ERK antisense/amphetamine (AMPH) co‐administration on the changes in hypothalamic pERK1/2, pCREB, NPY, POMC and MC 3 receptor (MC3R) expression during a 4 day period of drug treatment. Upper panel: each of the protein levels was measured by Western blot. Lower panel: the protein levels in drug‐treated groups were indicated as the percentage of the control group (Day 0). Each densitometric value represents the mean ± SEM; n = 6 for each group (total of seven drug‐treated groups). * P

    Article Snippet: Antibodies against NPY, tERK1/2, pERK1/2 and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot

    Suppression of MAP kinase signalling in miR-21 morphants and BDM-treated hearts. ( a – c ) In the WT beating hearts, a phosphorylated form of Erk1/2 (pErk1/2) was stained in the cuboidal endocardial cells at the AV junction (yellow arrowheads in a ). The entire endocardial cells were labelled by GFP, which expression was driven by the fli1 promoter ( b , c ). ( d – f ) In the miR-21 morphants, the pErk1/2 staining was week and the cells were flat. ( g – i ) When the heartbeat was stopped by BDM, phosphorylation of Erk1/2 was also suppressed, leaving only faint staining in the apical surface of flattened endocardial cells. ( j – o ) Injection of the miR-21 target protection MO and the U0126 treatment gave the same results on pErk1/s staining. A, atrium; V, ventricle. Scale bars, 20 μm.

    Journal: Nature Communications

    Article Title: Haemodynamically dependent valvulogenesis of zebrafish heart is mediated by flow-dependent expression of miR-21

    doi: 10.1038/ncomms2978

    Figure Lengend Snippet: Suppression of MAP kinase signalling in miR-21 morphants and BDM-treated hearts. ( a – c ) In the WT beating hearts, a phosphorylated form of Erk1/2 (pErk1/2) was stained in the cuboidal endocardial cells at the AV junction (yellow arrowheads in a ). The entire endocardial cells were labelled by GFP, which expression was driven by the fli1 promoter ( b , c ). ( d – f ) In the miR-21 morphants, the pErk1/2 staining was week and the cells were flat. ( g – i ) When the heartbeat was stopped by BDM, phosphorylation of Erk1/2 was also suppressed, leaving only faint staining in the apical surface of flattened endocardial cells. ( j – o ) Injection of the miR-21 target protection MO and the U0126 treatment gave the same results on pErk1/s staining. A, atrium; V, ventricle. Scale bars, 20 μm.

    Article Snippet: An antibody against pERK1/2 (1:100) was purchased from Cell Signaling Technology.

    Techniques: Staining, Expressing, Injection

    CRH-elicited cAMP response depends on bicarbonate. (A) HT22-CRHR1 cells were stimulated with CRH in HCO 3 − -free or 25 mM HCO 3 − DMEM. (B) HT22-CRHR1 cells preincubated with vehicle (control) or anion channel blocker DIDS were stimulated with CRH. pERK1/2 and total ERK1/2 were measured and expressed as the percentage of maximum pERK1/2 under control conditions (mean ± SEM, n = 3). *, P

    Journal: The Journal of Cell Biology

    Article Title: Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling

    doi: 10.1083/jcb.201512075

    Figure Lengend Snippet: CRH-elicited cAMP response depends on bicarbonate. (A) HT22-CRHR1 cells were stimulated with CRH in HCO 3 − -free or 25 mM HCO 3 − DMEM. (B) HT22-CRHR1 cells preincubated with vehicle (control) or anion channel blocker DIDS were stimulated with CRH. pERK1/2 and total ERK1/2 were measured and expressed as the percentage of maximum pERK1/2 under control conditions (mean ± SEM, n = 3). *, P

    Article Snippet: The following antibodies were used: anti-sAC (R21; CEP Biotech), anti–c-Myc (9E10; sc-40) and anti–pERK1/2 (E-4; sc-7383) from Santa Cruz Biotechnology, Inc.; anti–total-ERK1/2 (9102; Cell Signaling Technology), anti-phospho CREB (06-519; EMD Millipore), and anti–total-CREB (9104; Cell Signaling Technology).

    Techniques:

    sAC-generated cAMP has a specific role in CRHR1 signaling. (A–C) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells stimulated with CRH or isoproterenol for the indicated time points. Results are expressed as the percentage of maximum pERK1/2. (A) Cells were preincubated with vehicle (control) or sAC-specific (7.5 µM KH7) or tmAC-specific (100 µM ddA) inhibitors (mean ± SEM, n = 4). Two-way analysis of variance followed by Tukey’s test: *, P

    Journal: The Journal of Cell Biology

    Article Title: Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling

    doi: 10.1083/jcb.201512075

    Figure Lengend Snippet: sAC-generated cAMP has a specific role in CRHR1 signaling. (A–C) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells stimulated with CRH or isoproterenol for the indicated time points. Results are expressed as the percentage of maximum pERK1/2. (A) Cells were preincubated with vehicle (control) or sAC-specific (7.5 µM KH7) or tmAC-specific (100 µM ddA) inhibitors (mean ± SEM, n = 4). Two-way analysis of variance followed by Tukey’s test: *, P

    Article Snippet: The following antibodies were used: anti-sAC (R21; CEP Biotech), anti–c-Myc (9E10; sc-40) and anti–pERK1/2 (E-4; sc-7383) from Santa Cruz Biotechnology, Inc.; anti–total-ERK1/2 (9102; Cell Signaling Technology), anti-phospho CREB (06-519; EMD Millipore), and anti–total-CREB (9104; Cell Signaling Technology).

    Techniques: Generated

    CRH-triggered calcium response regulates cAMP. (A and B) HT22-CRHR1 cells cotransfected with Epac-S H187 and REX-GECO1 were stimulated with 100 nM CRH. (A) Representative images for each signal. Bar, 5 µm. (B) Time course of FRET changes (black trace) or REX-GECO1 fluorescence (orange trace). Data: mean ± SEM, 14 cells. (C) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells transfected with 50 nM siRNA against GL3 (control) or AC1 and stimulated with CRH. Results are expressed as the percentage of maximum pERK1/2 after stimulation under control conditions (mean ± SEM, n = 3). *, P

    Journal: The Journal of Cell Biology

    Article Title: Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling

    doi: 10.1083/jcb.201512075

    Figure Lengend Snippet: CRH-triggered calcium response regulates cAMP. (A and B) HT22-CRHR1 cells cotransfected with Epac-S H187 and REX-GECO1 were stimulated with 100 nM CRH. (A) Representative images for each signal. Bar, 5 µm. (B) Time course of FRET changes (black trace) or REX-GECO1 fluorescence (orange trace). Data: mean ± SEM, 14 cells. (C) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells transfected with 50 nM siRNA against GL3 (control) or AC1 and stimulated with CRH. Results are expressed as the percentage of maximum pERK1/2 after stimulation under control conditions (mean ± SEM, n = 3). *, P

    Article Snippet: The following antibodies were used: anti-sAC (R21; CEP Biotech), anti–c-Myc (9E10; sc-40) and anti–pERK1/2 (E-4; sc-7383) from Santa Cruz Biotechnology, Inc.; anti–total-ERK1/2 (9102; Cell Signaling Technology), anti-phospho CREB (06-519; EMD Millipore), and anti–total-CREB (9104; Cell Signaling Technology).

    Techniques: Fluorescence, Transfection

    CRH responses in AtT20 and 3T3L1-CRHR1 cells. (A) pERK1/2 and total ERK1/2 were measured in AtT20 cells preincubated with vehicle (control) or tmAC-specific (50 µM ddA) or sAC-specific (7.5 µM KH7 or 10 µM 2-HE) inhibitors and stimulated with CRH. (B) pERK1/2 and total ERK1/2 were measured in AtT20 cells transfected with 50 nM siRNA against GL3 (control) or sAC and stimulated with CRH or PACAP38. Results are expressed as the percentage of maximum pERK1/2 (mean ± SEM, n = 3). *, P

    Journal: The Journal of Cell Biology

    Article Title: Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling

    doi: 10.1083/jcb.201512075

    Figure Lengend Snippet: CRH responses in AtT20 and 3T3L1-CRHR1 cells. (A) pERK1/2 and total ERK1/2 were measured in AtT20 cells preincubated with vehicle (control) or tmAC-specific (50 µM ddA) or sAC-specific (7.5 µM KH7 or 10 µM 2-HE) inhibitors and stimulated with CRH. (B) pERK1/2 and total ERK1/2 were measured in AtT20 cells transfected with 50 nM siRNA against GL3 (control) or sAC and stimulated with CRH or PACAP38. Results are expressed as the percentage of maximum pERK1/2 (mean ± SEM, n = 3). *, P

    Article Snippet: The following antibodies were used: anti-sAC (R21; CEP Biotech), anti–c-Myc (9E10; sc-40) and anti–pERK1/2 (E-4; sc-7383) from Santa Cruz Biotechnology, Inc.; anti–total-ERK1/2 (9102; Cell Signaling Technology), anti-phospho CREB (06-519; EMD Millipore), and anti–total-CREB (9104; Cell Signaling Technology).

    Techniques: Transfection

    sAC effectors in HT22-CRHR1 and AtT20 cells. (A,B,D–F) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells stimulated with CRH for the indicated time points. (A, B, and D) Cells preincubated with vehicle (control), PKA inhibitors (A: 700 nM KT5720; B: 75 µM RpcAMPS), or EPAC-specific inhibitor (5 µM ESI09) were stimulated with CRH. In D, results are expressed as the percentage of maximum pERK1/2 after stimulation under control conditions (mean ± SEM, n = 3). *, P

    Journal: The Journal of Cell Biology

    Article Title: Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling

    doi: 10.1083/jcb.201512075

    Figure Lengend Snippet: sAC effectors in HT22-CRHR1 and AtT20 cells. (A,B,D–F) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells stimulated with CRH for the indicated time points. (A, B, and D) Cells preincubated with vehicle (control), PKA inhibitors (A: 700 nM KT5720; B: 75 µM RpcAMPS), or EPAC-specific inhibitor (5 µM ESI09) were stimulated with CRH. In D, results are expressed as the percentage of maximum pERK1/2 after stimulation under control conditions (mean ± SEM, n = 3). *, P

    Article Snippet: The following antibodies were used: anti-sAC (R21; CEP Biotech), anti–c-Myc (9E10; sc-40) and anti–pERK1/2 (E-4; sc-7383) from Santa Cruz Biotechnology, Inc.; anti–total-ERK1/2 (9102; Cell Signaling Technology), anti-phospho CREB (06-519; EMD Millipore), and anti–total-CREB (9104; Cell Signaling Technology).

    Techniques:

    sAC is critical for endosome-generated cAMP in response to CRH. (A) Schematic representation of the experimental design. HT22-CRHR1-Epac-S H187 cells transfected with pcDNA3 (control) or DynK44A plus mCherry for 48 h were pretreated with vehicle (DMSO) or tmAC-specific (100 µM ddA) or sAC-specific (20 µM 2-HE) inhibitors and stimulated with 10 nM CRH. (B) Time course of FRET changes was measured in single cells. The cAMP response is expressed as the percentage of maximum FRET signal obtained in each condition assayed after stimulation (mean ± SEM, n = 7). Student’s t test was performed for each time point. P values in gray; P = 0.05 is indicated with a dotted line. (C and D) Effect of 2-HE on agonist-induced CRHR1 internalization analyzed by fluorescence flow cytometry (C) and cMyc-GFP-CRHR1 subcellular distribution (D). HT22-CRHR1 cells preincubated with vehicle (control) or 20 µM 2-HE were stimulated with 10 nM CRH for the indicated times. (C) Fluorescence measured at time 0 was defined as 100% (mean ± SEM, n = 4, 10,000 cells/condition). (D) Representative images obtained for each condition. Arrowheads point to internalized GFP clusters. Bars, 5 µM. (E) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells preincubated with vehicle (control) or sAC-specific (7.5 µM KH7) or endocytosis-specific (30 µM Dyngo-4a) inhibitors and stimulated with CRH. Results are expressed as the percentage of maximum pERK1/2 (mean ± SEM, n = 4). Two-way ANOVA followed by Tukey’s test: *, P

    Journal: The Journal of Cell Biology

    Article Title: Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling

    doi: 10.1083/jcb.201512075

    Figure Lengend Snippet: sAC is critical for endosome-generated cAMP in response to CRH. (A) Schematic representation of the experimental design. HT22-CRHR1-Epac-S H187 cells transfected with pcDNA3 (control) or DynK44A plus mCherry for 48 h were pretreated with vehicle (DMSO) or tmAC-specific (100 µM ddA) or sAC-specific (20 µM 2-HE) inhibitors and stimulated with 10 nM CRH. (B) Time course of FRET changes was measured in single cells. The cAMP response is expressed as the percentage of maximum FRET signal obtained in each condition assayed after stimulation (mean ± SEM, n = 7). Student’s t test was performed for each time point. P values in gray; P = 0.05 is indicated with a dotted line. (C and D) Effect of 2-HE on agonist-induced CRHR1 internalization analyzed by fluorescence flow cytometry (C) and cMyc-GFP-CRHR1 subcellular distribution (D). HT22-CRHR1 cells preincubated with vehicle (control) or 20 µM 2-HE were stimulated with 10 nM CRH for the indicated times. (C) Fluorescence measured at time 0 was defined as 100% (mean ± SEM, n = 4, 10,000 cells/condition). (D) Representative images obtained for each condition. Arrowheads point to internalized GFP clusters. Bars, 5 µM. (E) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells preincubated with vehicle (control) or sAC-specific (7.5 µM KH7) or endocytosis-specific (30 µM Dyngo-4a) inhibitors and stimulated with CRH. Results are expressed as the percentage of maximum pERK1/2 (mean ± SEM, n = 4). Two-way ANOVA followed by Tukey’s test: *, P

    Article Snippet: The following antibodies were used: anti-sAC (R21; CEP Biotech), anti–c-Myc (9E10; sc-40) and anti–pERK1/2 (E-4; sc-7383) from Santa Cruz Biotechnology, Inc.; anti–total-ERK1/2 (9102; Cell Signaling Technology), anti-phospho CREB (06-519; EMD Millipore), and anti–total-CREB (9104; Cell Signaling Technology).

    Techniques: Generated, Transfection, Fluorescence, Flow Cytometry, Cytometry

    Glutamate injections upregulated pNR2B and pERK1/2 expression in the hippocampus. OVX rats were assigned to 5 groups for western blot analyses. At 5, 15, 30, and 45 min following the glutamate injections, rats were sacrificed and the intact hippocampus was dissected. In addition, in control group, we eliminated the possibility that the needle insertion produced protein expression changes by sacrificing rats immediately after vehicle injections. (A,B) Representative western blot of pNR2B and pERK1/2 after glutamate injections. Quantification of protein levels was normalized against loading control β-actin and presented as the relative density compared with the control group. * P

    Journal: Frontiers in Neurology

    Article Title: Genistein Antagonizes 17β-Estradiol Effects on Glutamate-Evoked Masseter Muscle Hypernociception in Rats

    doi: 10.3389/fneur.2018.00649

    Figure Lengend Snippet: Glutamate injections upregulated pNR2B and pERK1/2 expression in the hippocampus. OVX rats were assigned to 5 groups for western blot analyses. At 5, 15, 30, and 45 min following the glutamate injections, rats were sacrificed and the intact hippocampus was dissected. In addition, in control group, we eliminated the possibility that the needle insertion produced protein expression changes by sacrificing rats immediately after vehicle injections. (A,B) Representative western blot of pNR2B and pERK1/2 after glutamate injections. Quantification of protein levels was normalized against loading control β-actin and presented as the relative density compared with the control group. * P

    Article Snippet: After blocking with 5% non-fat milk for 1 h, the membranes were incubated with anti-pNR2B antibody (1:1,000; Cell Signaling Technology) or anti-pERK1/2 antibody (1:2,000; Cell Signaling Technology) overnight at 4°C.

    Techniques: Expressing, Western Blot, Produced

    Genistein blocked E2-induced pNR2B and pERK1/2 upregulation, and partially blocked E2-potentiated glutamate-evoked pNR2B and pERK1/2 upregulation in the hippocampus. OVX rats were assigned to 4 groups for western blot analyses: (1) no injections, (2) E2 replacement, (3) genistein treatment, and (4) genistein pretreatment combined with E2 replacement. (A,B) Representative western blots of pNR2B and pERK1/2 after E2 and genistein treatment. Quantification of protein levels was normalized against loading control β-actin and presented as the relative density compared with the control group (OVX). * P

    Journal: Frontiers in Neurology

    Article Title: Genistein Antagonizes 17β-Estradiol Effects on Glutamate-Evoked Masseter Muscle Hypernociception in Rats

    doi: 10.3389/fneur.2018.00649

    Figure Lengend Snippet: Genistein blocked E2-induced pNR2B and pERK1/2 upregulation, and partially blocked E2-potentiated glutamate-evoked pNR2B and pERK1/2 upregulation in the hippocampus. OVX rats were assigned to 4 groups for western blot analyses: (1) no injections, (2) E2 replacement, (3) genistein treatment, and (4) genistein pretreatment combined with E2 replacement. (A,B) Representative western blots of pNR2B and pERK1/2 after E2 and genistein treatment. Quantification of protein levels was normalized against loading control β-actin and presented as the relative density compared with the control group (OVX). * P

    Article Snippet: After blocking with 5% non-fat milk for 1 h, the membranes were incubated with anti-pNR2B antibody (1:1,000; Cell Signaling Technology) or anti-pERK1/2 antibody (1:2,000; Cell Signaling Technology) overnight at 4°C.

    Techniques: Western Blot

    E2 potentiated glutamate-evoked pNR2B and pERK1/2 upregulation in the hippocampus. OVX rats were assigned to 4 groups for western blot analyses: (1) no injections, (2) E2 replacement, (3) glutamate injections, and (4) E2 replacement combined with glutamate injections. (A, B) Representative western blots of pNR2B and pERK1/2 after E2 and glutamate treatment. Quantification of protein levels was normalized against loading control β-actin and presented as the relative density compared with the control group. * P

    Journal: Frontiers in Neurology

    Article Title: Genistein Antagonizes 17β-Estradiol Effects on Glutamate-Evoked Masseter Muscle Hypernociception in Rats

    doi: 10.3389/fneur.2018.00649

    Figure Lengend Snippet: E2 potentiated glutamate-evoked pNR2B and pERK1/2 upregulation in the hippocampus. OVX rats were assigned to 4 groups for western blot analyses: (1) no injections, (2) E2 replacement, (3) glutamate injections, and (4) E2 replacement combined with glutamate injections. (A, B) Representative western blots of pNR2B and pERK1/2 after E2 and glutamate treatment. Quantification of protein levels was normalized against loading control β-actin and presented as the relative density compared with the control group. * P

    Article Snippet: After blocking with 5% non-fat milk for 1 h, the membranes were incubated with anti-pNR2B antibody (1:1,000; Cell Signaling Technology) or anti-pERK1/2 antibody (1:2,000; Cell Signaling Technology) overnight at 4°C.

    Techniques: Western Blot

    Basal pERK1/2 signaling of mutant hMC3Rs. Results are expressed as percentage of WT basal pERK1/2 level. Shown are mean ± SEM of at least four experiments. Star (*) indicates significantly different from WT hMC3R, P

    Journal: International Journal of Biological Sciences

    Article Title: Biased Signaling in Naturally Occurring Mutations in Human Melanocortin-3 Receptor Gene

    doi: 10.7150/ijbs.11032

    Figure Lengend Snippet: Basal pERK1/2 signaling of mutant hMC3Rs. Results are expressed as percentage of WT basal pERK1/2 level. Shown are mean ± SEM of at least four experiments. Star (*) indicates significantly different from WT hMC3R, P

    Article Snippet: Approximately 24 h after transfection, cells were starved overnight, and then treated with either 10-5 M α-MSH or Waymouth/BSA alone for 5 min. Proteins were extracted in lysis buffer containing phosphatase and protease inhibitors, separated by 10% SDS-PAGE, and transferred onto PVDF membrane. pERK1/2 and β-tubulin levels were detected with primary rabbit anti-pERK1/2 antibody (1:1000 ~ 1:2000, Cell Signaling Technology, Beverly, MA) and mouse anti-β-tubulin antibody (1:5000 ~ 1:10,000, Developmental Studies Hybridoma Bank at University of Iowa, Iowa City, IA) in Tris-buffered saline with Tween-20 containing 5% BSA, respectively.

    Techniques: Mutagenesis

    Effects of LY3009120 on signaling and gene expression ( A ) CRC cell lines were treated with DMSO, 0.1 μM or 1 μM LY3009120 for 30 minutes ( left panel ) or 2 hrs ( right panel ). Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. ( B ) CRC cell lines were treated with increasing concentrations of LY3009120 and fixed at the times indicated. Cells were stained with Hoechst stain and nuclei counts were obtained. At the 72 hr time point, replicate plates were assessed for proliferation by CTG analysis. ( C ) CRC cell lines were treated with DMSO or logarithmic concentrations of LY3009120 and fixed at 24 hrs post-treatment. Cells were stained for immunofluorescence analysis with Hoechst stain and antibodies against the proteins indicated and the average intensity of the signal for each analyte was measured by HCI. Results are plotted as percent of DMSO-treated cells and are representative of two independent experiments. The signal for pERK1/2 T202/Y204 and pS6 S240/244 was normalized to the levels of ERK1/2 and S6 respectively. ( D ) CRC cell lines were treated with DMSO or 1 μM LY3009120 for 24 and 48 hrs. Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. ( E ) CRC cell lines were treated with DMSO or increasing concentrations of LY3009120 for 24 hrs. Expression levels of the indicated genes were assessed by Affymetrix analysis.

    Journal: Oncotarget

    Article Title: LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer

    doi: 10.18632/oncotarget.14002

    Figure Lengend Snippet: Effects of LY3009120 on signaling and gene expression ( A ) CRC cell lines were treated with DMSO, 0.1 μM or 1 μM LY3009120 for 30 minutes ( left panel ) or 2 hrs ( right panel ). Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. ( B ) CRC cell lines were treated with increasing concentrations of LY3009120 and fixed at the times indicated. Cells were stained with Hoechst stain and nuclei counts were obtained. At the 72 hr time point, replicate plates were assessed for proliferation by CTG analysis. ( C ) CRC cell lines were treated with DMSO or logarithmic concentrations of LY3009120 and fixed at 24 hrs post-treatment. Cells were stained for immunofluorescence analysis with Hoechst stain and antibodies against the proteins indicated and the average intensity of the signal for each analyte was measured by HCI. Results are plotted as percent of DMSO-treated cells and are representative of two independent experiments. The signal for pERK1/2 T202/Y204 and pS6 S240/244 was normalized to the levels of ERK1/2 and S6 respectively. ( D ) CRC cell lines were treated with DMSO or 1 μM LY3009120 for 24 and 48 hrs. Whole cell lysates were analyzed by Western blot using antibodies against the proteins indicated. ( E ) CRC cell lines were treated with DMSO or increasing concentrations of LY3009120 for 24 hrs. Expression levels of the indicated genes were assessed by Affymetrix analysis.

    Article Snippet: Objects were identified using an algorithm to detect nuclear staining with Hoechst dye, and the relative levels of cleaved caspase 3 (Cell Signaling #9661), cleaved PARP (Cell Signaling #5625), pERK1/2 T202/Y204 (Cell Signaling #4370), pS6 S240/244 (Cell Signaling #5364), and p27 (Cell Signaling #3686) (all rabbit) and pHH3 (Cell Signaling #9706), S6 (Cell Signaling #2317), and ERK1/2 (Cell Signaling #4696) (all mouse) were determined through the intensities of Alexafluor-647 goat anti-rabbit (Invitrogen #A21245) and Alexafluor-555 goat anti-mouse (Invitrogen #A21425) respectively.

    Techniques: Expressing, Western Blot, Staining, CTG Assay, Immunofluorescence

    Effects of LY3009120 on cell cycle and apoptosis ( A ) Representative experiment of CRC cell lines treated with either DMSO or LY3009120 (0.5 μM) for the times indicated, fixed and stained with propidium iodide and analyzed for cell cycle by flow cytometry. ( B ) CRC cell lines were treated with increasing concentrations of LY3009120, fixed at 24 or 48 hrs ( left and right panels respectively) and stained for immunofluorescence with Click-iT ® EdU and antibodies against pERK1/2 T202/Y204 and pHH3 S10 as indicated. The average intensity of the signal for each analyte was measured by HCI. The data are representative of two independent experiments each conducted in triplicate technical replicates, with results plotted as percent of DMSO-treated cells. ( C ) Cells were treated with increasing concentrations of LY3009120 and fixed at 48 hrs post-treatment. Cells were stained for immunofluorescence analysis with antibodies against the proteins indicated and the average intensity of the signal for each analyte was measured by HCI. Results are plotted as percent of DMSO-treated cells and are representative of two independent experiments.

    Journal: Oncotarget

    Article Title: LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer

    doi: 10.18632/oncotarget.14002

    Figure Lengend Snippet: Effects of LY3009120 on cell cycle and apoptosis ( A ) Representative experiment of CRC cell lines treated with either DMSO or LY3009120 (0.5 μM) for the times indicated, fixed and stained with propidium iodide and analyzed for cell cycle by flow cytometry. ( B ) CRC cell lines were treated with increasing concentrations of LY3009120, fixed at 24 or 48 hrs ( left and right panels respectively) and stained for immunofluorescence with Click-iT ® EdU and antibodies against pERK1/2 T202/Y204 and pHH3 S10 as indicated. The average intensity of the signal for each analyte was measured by HCI. The data are representative of two independent experiments each conducted in triplicate technical replicates, with results plotted as percent of DMSO-treated cells. ( C ) Cells were treated with increasing concentrations of LY3009120 and fixed at 48 hrs post-treatment. Cells were stained for immunofluorescence analysis with antibodies against the proteins indicated and the average intensity of the signal for each analyte was measured by HCI. Results are plotted as percent of DMSO-treated cells and are representative of two independent experiments.

    Article Snippet: Objects were identified using an algorithm to detect nuclear staining with Hoechst dye, and the relative levels of cleaved caspase 3 (Cell Signaling #9661), cleaved PARP (Cell Signaling #5625), pERK1/2 T202/Y204 (Cell Signaling #4370), pS6 S240/244 (Cell Signaling #5364), and p27 (Cell Signaling #3686) (all rabbit) and pHH3 (Cell Signaling #9706), S6 (Cell Signaling #2317), and ERK1/2 (Cell Signaling #4696) (all mouse) were determined through the intensities of Alexafluor-647 goat anti-rabbit (Invitrogen #A21245) and Alexafluor-555 goat anti-mouse (Invitrogen #A21425) respectively.

    Techniques: Staining, Flow Cytometry, Cytometry, Immunofluorescence

    L-NAME inhibits NR1 and ERK1/2 phosphorylation, as well as nNOS and iNOS upregulation in db/db mice. A: Representative immunoblots of pNR1, total NR1, and actin from the LSC of db+ and db/db mice intrathecally treated with control aCSF (C) or aCSF with L-NAME. L-NAME treatment decreased NR1 phosphorylation in db/db, but not db+ mice. B: Densitometric analysis of pNR1 immunoblots. L-NAME treatment decreased the levels of NR1 phosphorylation back to that of control db+ mice. C: Representative immunoblots of pERK1/2, ERK1/2, and actin from the LSC of db+ and db/db mice treated with control solution (C) or L-NAME. L-NAME treatment decreased ERK1/2 phosphorylation in db/db, but not db+ mice. D: Densitometric analysis of pERK1/2 immunoblots. L-NAME treatment decreased the levels of ERK1/2 phosphorylation back to control levels. E: nNOS immunoblots revealed that L-NAME treatment reduced nNOS upregulation in db/db mice. F: Densitometric analysis of nNOS immunoblots demonstrated L-NAME treatment decreased nNOS levels in db/db mice back to control levels. G: iNOS immunoblots showed that L-NAME treatment reduced iNOS upregulation in db/db mice. H: Densitometric analysis of iNOS immunoblots demonstrated iNOS treatment decreased iNOS levels in db/db mice back to control levels. **, p

    Journal: Glia

    Article Title: Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

    doi: 10.1002/glia.22349

    Figure Lengend Snippet: L-NAME inhibits NR1 and ERK1/2 phosphorylation, as well as nNOS and iNOS upregulation in db/db mice. A: Representative immunoblots of pNR1, total NR1, and actin from the LSC of db+ and db/db mice intrathecally treated with control aCSF (C) or aCSF with L-NAME. L-NAME treatment decreased NR1 phosphorylation in db/db, but not db+ mice. B: Densitometric analysis of pNR1 immunoblots. L-NAME treatment decreased the levels of NR1 phosphorylation back to that of control db+ mice. C: Representative immunoblots of pERK1/2, ERK1/2, and actin from the LSC of db+ and db/db mice treated with control solution (C) or L-NAME. L-NAME treatment decreased ERK1/2 phosphorylation in db/db, but not db+ mice. D: Densitometric analysis of pERK1/2 immunoblots. L-NAME treatment decreased the levels of ERK1/2 phosphorylation back to control levels. E: nNOS immunoblots revealed that L-NAME treatment reduced nNOS upregulation in db/db mice. F: Densitometric analysis of nNOS immunoblots demonstrated L-NAME treatment decreased nNOS levels in db/db mice back to control levels. G: iNOS immunoblots showed that L-NAME treatment reduced iNOS upregulation in db/db mice. H: Densitometric analysis of iNOS immunoblots demonstrated iNOS treatment decreased iNOS levels in db/db mice back to control levels. **, p

    Article Snippet: Sections were then incubated at room temperature for 16-24 h with primary antibodies: pERK1/2 (1:100), substance P (1:500, rat monoclonal, Abcam), nNOS (1:200), iNOS (1:100), and GFAP (1:1000, Abcam).

    Techniques: Mouse Assay, Western Blot

    UO126 has no effect on nNOS and iNOS expression. A: Representative immunoblots of pERK1/2, total ERK1/2, and actin from the LSC of db+ and db/db mice intrathecally treated with control aCSF (C) or aCSF with UO126. UO126 treatment decreased ERK1/2 phosphorylation in db/db, but not db+ mice. B: Densitometric analysis of pERK1/2 immunoblots. UO126 treatment decreased the levels of ERK1/2 phosphorylation back to control levels. C: Representative immunoblots of nNOS, iNOS, and actin from the LSC of db+ and db/db mice treated with control solution (C) or UO126. UO126 treatment had no effect on nNOS and iNOS levels in db/db mice. D,E: Densitometric analysis revealed UO126 treatment did not affect nNOS and iNOS levels in db/db mice. **, p

    Journal: Glia

    Article Title: Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

    doi: 10.1002/glia.22349

    Figure Lengend Snippet: UO126 has no effect on nNOS and iNOS expression. A: Representative immunoblots of pERK1/2, total ERK1/2, and actin from the LSC of db+ and db/db mice intrathecally treated with control aCSF (C) or aCSF with UO126. UO126 treatment decreased ERK1/2 phosphorylation in db/db, but not db+ mice. B: Densitometric analysis of pERK1/2 immunoblots. UO126 treatment decreased the levels of ERK1/2 phosphorylation back to control levels. C: Representative immunoblots of nNOS, iNOS, and actin from the LSC of db+ and db/db mice treated with control solution (C) or UO126. UO126 treatment had no effect on nNOS and iNOS levels in db/db mice. D,E: Densitometric analysis revealed UO126 treatment did not affect nNOS and iNOS levels in db/db mice. **, p

    Article Snippet: Sections were then incubated at room temperature for 16-24 h with primary antibodies: pERK1/2 (1:100), substance P (1:500, rat monoclonal, Abcam), nNOS (1:200), iNOS (1:100), and GFAP (1:1000, Abcam).

    Techniques: Expressing, Western Blot, Mouse Assay

    MK801 treatment significantly reduces the numbers pERK1/2 and nNOS-positive neurons in the LSCDH of db/db mice. A, D: MK801 treatment had no effect on SP expression in the LSCDH of db/db mice. B, E: MK801 treatment reduced the number of pERK1/2-positive neurons in the LSCDH of db/db mice. C, F: MK801 treatment reduced the number of nNOS-positive neurons in the LSCDH of db/db mice. G: MK801 treatment reduced the number of pERK1/2-positive neurons in the LSCDH in db/db mice back to control levels. H: MK801 treatment reduced the numbers of nNOS-positive neurons in the LSCDH in db/db mice back to control levels. **, p

    Journal: Glia

    Article Title: Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

    doi: 10.1002/glia.22349

    Figure Lengend Snippet: MK801 treatment significantly reduces the numbers pERK1/2 and nNOS-positive neurons in the LSCDH of db/db mice. A, D: MK801 treatment had no effect on SP expression in the LSCDH of db/db mice. B, E: MK801 treatment reduced the number of pERK1/2-positive neurons in the LSCDH of db/db mice. C, F: MK801 treatment reduced the number of nNOS-positive neurons in the LSCDH of db/db mice. G: MK801 treatment reduced the number of pERK1/2-positive neurons in the LSCDH in db/db mice back to control levels. H: MK801 treatment reduced the numbers of nNOS-positive neurons in the LSCDH in db/db mice back to control levels. **, p

    Article Snippet: Sections were then incubated at room temperature for 16-24 h with primary antibodies: pERK1/2 (1:100), substance P (1:500, rat monoclonal, Abcam), nNOS (1:200), iNOS (1:100), and GFAP (1:1000, Abcam).

    Techniques: Mouse Assay, Expressing

    Increased pERK1/2-positive neurons in the LSCDH of db/db mice. A-C: SP (A) and pERK1/2 (B) immunohistochemistry in db+ mice. Minimal pERK1/2 positive neurons were detected in db+ mice (B, arrow). D-F: SP (D) and pERK1/2 (E) immunohistochemistry in db/db mice. In comparison to db+ mice, an increase in pERK1/2-positive neurons was observed in lamina I-III of db/db mice (E, arrows). Bar = 20 μm. N = 4.

    Journal: Glia

    Article Title: Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

    doi: 10.1002/glia.22349

    Figure Lengend Snippet: Increased pERK1/2-positive neurons in the LSCDH of db/db mice. A-C: SP (A) and pERK1/2 (B) immunohistochemistry in db+ mice. Minimal pERK1/2 positive neurons were detected in db+ mice (B, arrow). D-F: SP (D) and pERK1/2 (E) immunohistochemistry in db/db mice. In comparison to db+ mice, an increase in pERK1/2-positive neurons was observed in lamina I-III of db/db mice (E, arrows). Bar = 20 μm. N = 4.

    Article Snippet: Sections were then incubated at room temperature for 16-24 h with primary antibodies: pERK1/2 (1:100), substance P (1:500, rat monoclonal, Abcam), nNOS (1:200), iNOS (1:100), and GFAP (1:1000, Abcam).

    Techniques: Mouse Assay, Immunohistochemistry

    Increased phosphorylation of NR1 and ERK1/2, but not JNK in the LSC of db/db mice. A: Representative immunoblots for pNR1 and total NR1. Increased NR1 phosphorylation was detected from 8-16 wk of age with maximal pNR1 levels detected at 10 wk of age. B: Densitometric analysis of NR1 phosphorylation. A 2.5-fold increase in NR1 phosphorylation was detected at 10 wk of age. C: Representative immunoblots for pERK1/2 and total ERK1/2. Increased ERK1/2 phosphorylation was detected from 8-16 wk of age with maximum pERK1/2 levels detected at 10 wk of age. D: Densitometric analysis of ERK1/2 phosphorylation. A 3-fold increase in ERK1/2 phosphorylation was detected at 10 wk of age. E: Representative immunoblots for pJNK and total JNK. F: Densitometric analysis of JNK phosphorylation. No significant change of JNK phosphorylation was detected in db/db mice. *, p

    Journal: Glia

    Article Title: Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

    doi: 10.1002/glia.22349

    Figure Lengend Snippet: Increased phosphorylation of NR1 and ERK1/2, but not JNK in the LSC of db/db mice. A: Representative immunoblots for pNR1 and total NR1. Increased NR1 phosphorylation was detected from 8-16 wk of age with maximal pNR1 levels detected at 10 wk of age. B: Densitometric analysis of NR1 phosphorylation. A 2.5-fold increase in NR1 phosphorylation was detected at 10 wk of age. C: Representative immunoblots for pERK1/2 and total ERK1/2. Increased ERK1/2 phosphorylation was detected from 8-16 wk of age with maximum pERK1/2 levels detected at 10 wk of age. D: Densitometric analysis of ERK1/2 phosphorylation. A 3-fold increase in ERK1/2 phosphorylation was detected at 10 wk of age. E: Representative immunoblots for pJNK and total JNK. F: Densitometric analysis of JNK phosphorylation. No significant change of JNK phosphorylation was detected in db/db mice. *, p

    Article Snippet: Sections were then incubated at room temperature for 16-24 h with primary antibodies: pERK1/2 (1:100), substance P (1:500, rat monoclonal, Abcam), nNOS (1:200), iNOS (1:100), and GFAP (1:1000, Abcam).

    Techniques: Mouse Assay, Western Blot

    MK801 inhibits NR1 and ERK1/2 phosphorylation, as well as nNOS and iNOS upregulation in db/db mice. A: Representative immunoblots of pNR1, total NR1, and actin from the LSC of db+ and db/db mice treated with control solution (C) or MK801. MK801 treatment decreased NR1 phosphorylation in db/db LSC, and had no effect on db+ mice. B: Densitometric analysis of pNR1 immunoblots. MK801 treatment decreased levels of NR1 phosphorylation back to control levels. C: Representative immunoblots of pERK1/2, total ERK1/2, and actin from LSC of db+ and db/db mice treated with control solution (C) or MK801. MK801 treatment decreased ERK1/2 phosphorylation in db/db but not in db+ mice. D: Densitometric analysis of pERK1/2 immunoblots. MK801 treatment decreased the levels of ERK1/2 phosphorylation back to control levels. E: nNOS immunoblots revealed that MK801 treatment reduced nNOS upregulation in db/db mice. F: Densitometric analysis of nNOS immunoblots demonstrated MK801 treatment decreased nNOS levels in db/db mice back to control levels. G: iNOS immunoblots showed that MK801 treatment reduced iNOS upregulation in db/db mice. H: Densitometric analysis of iNOS immunoblots demonstrated MK801 treatment decreased iNOS levels in db/db mice back to control levels. **, p

    Journal: Glia

    Article Title: Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

    doi: 10.1002/glia.22349

    Figure Lengend Snippet: MK801 inhibits NR1 and ERK1/2 phosphorylation, as well as nNOS and iNOS upregulation in db/db mice. A: Representative immunoblots of pNR1, total NR1, and actin from the LSC of db+ and db/db mice treated with control solution (C) or MK801. MK801 treatment decreased NR1 phosphorylation in db/db LSC, and had no effect on db+ mice. B: Densitometric analysis of pNR1 immunoblots. MK801 treatment decreased levels of NR1 phosphorylation back to control levels. C: Representative immunoblots of pERK1/2, total ERK1/2, and actin from LSC of db+ and db/db mice treated with control solution (C) or MK801. MK801 treatment decreased ERK1/2 phosphorylation in db/db but not in db+ mice. D: Densitometric analysis of pERK1/2 immunoblots. MK801 treatment decreased the levels of ERK1/2 phosphorylation back to control levels. E: nNOS immunoblots revealed that MK801 treatment reduced nNOS upregulation in db/db mice. F: Densitometric analysis of nNOS immunoblots demonstrated MK801 treatment decreased nNOS levels in db/db mice back to control levels. G: iNOS immunoblots showed that MK801 treatment reduced iNOS upregulation in db/db mice. H: Densitometric analysis of iNOS immunoblots demonstrated MK801 treatment decreased iNOS levels in db/db mice back to control levels. **, p

    Article Snippet: Sections were then incubated at room temperature for 16-24 h with primary antibodies: pERK1/2 (1:100), substance P (1:500, rat monoclonal, Abcam), nNOS (1:200), iNOS (1:100), and GFAP (1:1000, Abcam).

    Techniques: Mouse Assay, Western Blot

    Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p

    Journal: Frontiers in Immunology

    Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling

    doi: 10.3389/fimmu.2017.00265

    Figure Lengend Snippet: Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p

    Article Snippet: TCR Signaling Analysis by Flow Cytometry Infant CTRL and MAT T cells (5 × 105 cells/50 μl) that were freshly isolated (unstimulated) or stimulated with anti-CD3/anti-CD28 for 24, 48, and 72 h were incubated with or without soluble anti-CD3 (10 μg/ml; clone 145-2C11; BioLegend) and soluble anti-CD28 (10 μg/ml; clone 37.51, BioLegend) in cold RPMI 1640 supplemented with 0.5% FBS in 96 well round bottom plates at 4°C for 15 min, washed with cold medium, incubated with or without soluble goat anti-hamster IgG (20 μg/ml; Jackson ImmunoResearch Laboratories) at 4°C for 15 min, washed with cold medium, then resuspended in 50 μl of medium and incubated in a 37°C water bath for 2 min. After CD3/CD28 crosslinking, cells were immediately fixed with 50 μl of pre-warmed Cytofix buffer (BD Biosciences) for Erk2 and pErk1/2 staining or IC Fixation buffer (eBioscience) for ZAP-70, pZAP-70, pTyr, and c-Rel staining, incubated at 37°C for 10 min, and washed with FACS buffer (HBSS containing 1% FBS and 0.1% sodium azide) prior to staining with antibodies for CD8α, CD44, and CD62L at 4°C for 30 min. For intracellular staining of Erk2 and pErk1/2, cells were permeabilized with pre-chilled Phosflow Perm III buffer (BD Biosciences).

    Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Negative Staining, Fluorescence, Two Tailed Test

    Maternal antibiotic treatment (MAT) effector CD8 + T cells do not sustain phosphorylation of Erk-1/2 . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were stimulated with anti-CD3/anti-CD28 for 24, 48, and 72 h and analyzed for (A) percentage of Erk2 + cells (CTRL, n = 7; MAT, n = 6), (B) expression of Erk2 (CTRL, n = 7; MAT, n = 6), (C) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 7; MAT, n = 6), and (D) expression of pErk1/2 gating on effector (CD44 + ) CD8 + T cells (Teff). (E) Percentage of CD8 + Teff cells expressing low or high levels of Erk2 (Erk2 lo and Erk2 hi , respectively) and gating analysis of their corresponding pErk1/2-expressing populations at 48 h post-stimulation. Data are representative of two independent experiments and presented as mean + SEM. * p

    Journal: Frontiers in Immunology

    Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling

    doi: 10.3389/fimmu.2017.00265

    Figure Lengend Snippet: Maternal antibiotic treatment (MAT) effector CD8 + T cells do not sustain phosphorylation of Erk-1/2 . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were stimulated with anti-CD3/anti-CD28 for 24, 48, and 72 h and analyzed for (A) percentage of Erk2 + cells (CTRL, n = 7; MAT, n = 6), (B) expression of Erk2 (CTRL, n = 7; MAT, n = 6), (C) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 7; MAT, n = 6), and (D) expression of pErk1/2 gating on effector (CD44 + ) CD8 + T cells (Teff). (E) Percentage of CD8 + Teff cells expressing low or high levels of Erk2 (Erk2 lo and Erk2 hi , respectively) and gating analysis of their corresponding pErk1/2-expressing populations at 48 h post-stimulation. Data are representative of two independent experiments and presented as mean + SEM. * p

    Article Snippet: TCR Signaling Analysis by Flow Cytometry Infant CTRL and MAT T cells (5 × 105 cells/50 μl) that were freshly isolated (unstimulated) or stimulated with anti-CD3/anti-CD28 for 24, 48, and 72 h were incubated with or without soluble anti-CD3 (10 μg/ml; clone 145-2C11; BioLegend) and soluble anti-CD28 (10 μg/ml; clone 37.51, BioLegend) in cold RPMI 1640 supplemented with 0.5% FBS in 96 well round bottom plates at 4°C for 15 min, washed with cold medium, incubated with or without soluble goat anti-hamster IgG (20 μg/ml; Jackson ImmunoResearch Laboratories) at 4°C for 15 min, washed with cold medium, then resuspended in 50 μl of medium and incubated in a 37°C water bath for 2 min. After CD3/CD28 crosslinking, cells were immediately fixed with 50 μl of pre-warmed Cytofix buffer (BD Biosciences) for Erk2 and pErk1/2 staining or IC Fixation buffer (eBioscience) for ZAP-70, pZAP-70, pTyr, and c-Rel staining, incubated at 37°C for 10 min, and washed with FACS buffer (HBSS containing 1% FBS and 0.1% sodium azide) prior to staining with antibodies for CD8α, CD44, and CD62L at 4°C for 30 min. For intracellular staining of Erk2 and pErk1/2, cells were permeabilized with pre-chilled Phosflow Perm III buffer (BD Biosciences).

    Techniques: Mouse Assay, Expressing

    Acute restraint stress prevents formalin-induced increases in pERK2 in the CeA of male mice Following restraint (or control habituation), male mice received an injection of either 2% formalin (n=6) or sterile saline (n=6) in the rear right paw. Three hours following injection, animals were sacrificed and CeA tissue was isolated. Using Western blotting techniques, ( A. ) the CeA was assessed for expression of pERK1/2, well characterized pain signaling molecules, and total ERK1/2. B. Formalin treatment caused a significant increase in pERK1 (Two-way ANOVA: effect of formalin, p

    Journal: Physiology & behavior

    Article Title: Hormonal and molecular effects of restraint stress on formalin-induced pain-like behavior in male and female mice

    doi: 10.1016/j.physbeh.2016.08.009

    Figure Lengend Snippet: Acute restraint stress prevents formalin-induced increases in pERK2 in the CeA of male mice Following restraint (or control habituation), male mice received an injection of either 2% formalin (n=6) or sterile saline (n=6) in the rear right paw. Three hours following injection, animals were sacrificed and CeA tissue was isolated. Using Western blotting techniques, ( A. ) the CeA was assessed for expression of pERK1/2, well characterized pain signaling molecules, and total ERK1/2. B. Formalin treatment caused a significant increase in pERK1 (Two-way ANOVA: effect of formalin, p

    Article Snippet: Membranes were incubated in Odyssey blocking buffer for one hour and then incubated with mouse anti-pERK1/2 (Cell Signaling, 1:1,000) and rabbit anti-ERK1/2 (Cell Signaling, 1:1,000) primary antibodies for one hour.

    Techniques: Mouse Assay, Injection, Isolation, Western Blot, Expressing

    Formalin injection fails to increase pERK2 expression in the CeA of female mice Following restraint (or control habituation), female mice received an injection of either 2% formalin (n=12) or sterile saline (n=12) in the rear right paw. Three hours following injections, animals were sacrificed and CeA tissue was isolated. Using Western blotting ( A. ), the CeA was assessed for expression of pERK1/2 and ERK1/2. B. Similar to male mice, female mice exhibited formalin-dependent increases in pERK1 that were not blocked by acute restraint stress (Two-way ANOVA: effect of formalin, p=0.019; restraint, p > 0.05). C . Unlike their male counterparts however, female mice failed to demonstrate increases in pERK2 following peripheral inflammation (Two-way ANOVA: effect of formalin and restraint, p > 0.05).

    Journal: Physiology & behavior

    Article Title: Hormonal and molecular effects of restraint stress on formalin-induced pain-like behavior in male and female mice

    doi: 10.1016/j.physbeh.2016.08.009

    Figure Lengend Snippet: Formalin injection fails to increase pERK2 expression in the CeA of female mice Following restraint (or control habituation), female mice received an injection of either 2% formalin (n=12) or sterile saline (n=12) in the rear right paw. Three hours following injections, animals were sacrificed and CeA tissue was isolated. Using Western blotting ( A. ), the CeA was assessed for expression of pERK1/2 and ERK1/2. B. Similar to male mice, female mice exhibited formalin-dependent increases in pERK1 that were not blocked by acute restraint stress (Two-way ANOVA: effect of formalin, p=0.019; restraint, p > 0.05). C . Unlike their male counterparts however, female mice failed to demonstrate increases in pERK2 following peripheral inflammation (Two-way ANOVA: effect of formalin and restraint, p > 0.05).

    Article Snippet: Membranes were incubated in Odyssey blocking buffer for one hour and then incubated with mouse anti-pERK1/2 (Cell Signaling, 1:1,000) and rabbit anti-ERK1/2 (Cell Signaling, 1:1,000) primary antibodies for one hour.

    Techniques: Injection, Expressing, Mouse Assay, Isolation, Western Blot

    Dose-response effect of Ang-(1–7) on ERK1/2 phosphorylation levels. HEK293T cells were transfected either with the pcDNA3.1/myc-MasR vector or with the empty vector (pcDNA 3.1), starved in DMEM without serum for 8 h and then incubated for 5 min with increasing concentrations of Ang-(1–7). (A) Phosphorylation levels of ERK1/2 at activating residues Thr202/Tyr204, total ERK1/2 and β-tubulin abundance were determined by Western Blotting. (B) Data quantification. Bands intensity was quantified by optical densitometry, each individual value of pERK was normalized to that of total ERK and expressed as relative to the value obtained after incubation with cells transfected with pcDNA 3.1 and incubated with vehicle (basal value). Data are means ± SEM of three independent experiments. Values were fitted to a sigmoidal dose-response curve. Maximal response best-fitted values were analyzed by extra sum-of-squares F test, significantly different ( P

    Journal: Frontiers in Pharmacology

    Article Title: Participation of Gαi-Adenylate Cyclase and ERK1/2 in Mas Receptor Signaling Pathways

    doi: 10.3389/fphar.2019.00146

    Figure Lengend Snippet: Dose-response effect of Ang-(1–7) on ERK1/2 phosphorylation levels. HEK293T cells were transfected either with the pcDNA3.1/myc-MasR vector or with the empty vector (pcDNA 3.1), starved in DMEM without serum for 8 h and then incubated for 5 min with increasing concentrations of Ang-(1–7). (A) Phosphorylation levels of ERK1/2 at activating residues Thr202/Tyr204, total ERK1/2 and β-tubulin abundance were determined by Western Blotting. (B) Data quantification. Bands intensity was quantified by optical densitometry, each individual value of pERK was normalized to that of total ERK and expressed as relative to the value obtained after incubation with cells transfected with pcDNA 3.1 and incubated with vehicle (basal value). Data are means ± SEM of three independent experiments. Values were fitted to a sigmoidal dose-response curve. Maximal response best-fitted values were analyzed by extra sum-of-squares F test, significantly different ( P

    Article Snippet: Total cell lysates were resolved by SDS-PAGE, blotted and incubated with primary antibodies anti-myc (cat# 2272), anti-pERK1/2 (Thr202/Tyr204) (cat# 9101), anti-ERK1/2 (cat# 9102) (Cell Signaling Technology, Beverly, MA, United States) or anti-βtubulin (cat# 6046) (Abcam, Cambridge, MA, United States).

    Techniques: Transfection, Plasmid Preparation, Incubation, Western Blot

    Participation of the MasR and AT1R in Ang-(1–7) induced ERK phosphorylation. (A) Levels of mRNA coding for AT1R and MasR in HEK293T cells. The levels of mRNA were determined by qPCR in HEK293T cells transfected with pcDNA 3.1 and pcDNA 3.1/myc-MasR vectors as described in Section “Materials and Methods.” Data represent the mean ± SEM of three independent experiments. (B) Levels of mRNA coding for AT1R in cells transfected with AT1R siRNA. HEK293T cells were transfected with scrambled siRNA or AT1R siRNA and after 48 h the mRNA levels coding for AT1R were determined by qPCR as described in Section “Materials and Methods.” Data represent the mean ± SEM of three independent experiments. (C) Effect of silencing the AT1R on Ang-(1–7)-induced activation of ERK1/2. HEK293T cells that were transfected with the pcDNA3.1/myc-MasR plasmid or with the empty vector (pcDNA 3.1), were incubated for 5 min with Ang-(1–7) at the indicated concentrations. Experiments were performed in AT1R-silenced cells and in control cells (scrambled siRNA). Phosphorylation levels of ERK1/2 at activating Thr202/Tyr204, total ERK1/2 and β-tubulin abundance were determined by Western Blotting. Bands intensities were quantified by optical densitometry, pERK individual values were normalized to total ERK1/2 values and expressed as relative to the value obtained with control cells (transfected with pcDNA 3.1 and scramble siRNA) and incubated with vehicle (basal value). Data are means ± SEM of three independent experiments. Different letters denote significant difference at P

    Journal: Frontiers in Pharmacology

    Article Title: Participation of Gαi-Adenylate Cyclase and ERK1/2 in Mas Receptor Signaling Pathways

    doi: 10.3389/fphar.2019.00146

    Figure Lengend Snippet: Participation of the MasR and AT1R in Ang-(1–7) induced ERK phosphorylation. (A) Levels of mRNA coding for AT1R and MasR in HEK293T cells. The levels of mRNA were determined by qPCR in HEK293T cells transfected with pcDNA 3.1 and pcDNA 3.1/myc-MasR vectors as described in Section “Materials and Methods.” Data represent the mean ± SEM of three independent experiments. (B) Levels of mRNA coding for AT1R in cells transfected with AT1R siRNA. HEK293T cells were transfected with scrambled siRNA or AT1R siRNA and after 48 h the mRNA levels coding for AT1R were determined by qPCR as described in Section “Materials and Methods.” Data represent the mean ± SEM of three independent experiments. (C) Effect of silencing the AT1R on Ang-(1–7)-induced activation of ERK1/2. HEK293T cells that were transfected with the pcDNA3.1/myc-MasR plasmid or with the empty vector (pcDNA 3.1), were incubated for 5 min with Ang-(1–7) at the indicated concentrations. Experiments were performed in AT1R-silenced cells and in control cells (scrambled siRNA). Phosphorylation levels of ERK1/2 at activating Thr202/Tyr204, total ERK1/2 and β-tubulin abundance were determined by Western Blotting. Bands intensities were quantified by optical densitometry, pERK individual values were normalized to total ERK1/2 values and expressed as relative to the value obtained with control cells (transfected with pcDNA 3.1 and scramble siRNA) and incubated with vehicle (basal value). Data are means ± SEM of three independent experiments. Different letters denote significant difference at P

    Article Snippet: Total cell lysates were resolved by SDS-PAGE, blotted and incubated with primary antibodies anti-myc (cat# 2272), anti-pERK1/2 (Thr202/Tyr204) (cat# 9101), anti-ERK1/2 (cat# 9102) (Cell Signaling Technology, Beverly, MA, United States) or anti-βtubulin (cat# 6046) (Abcam, Cambridge, MA, United States).

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Activation Assay, Plasmid Preparation, Incubation, Western Blot

    Effect of zileuton and montelukast on ERK1/2 activation. Western blot analysis of extracts of spinal cord tissue collected at 24 h after injury. SCI caused an increase of the ERK1/2 phosphorylation in vehicle-treated mice. The treatment with zileuton or montelukast reduced pERK1/2 levels. Densitometric analysis of protein expression represents the mean±s.e.mean of 10 spinal cord tissues. Data were normalized on the basis of ERK-2 levels. ° P

    Journal: British Journal of Pharmacology

    Article Title: Effects of zileuton and montelukast in mouse experimental spinal cord injury

    doi: 10.1038/sj.bjp.0707577

    Figure Lengend Snippet: Effect of zileuton and montelukast on ERK1/2 activation. Western blot analysis of extracts of spinal cord tissue collected at 24 h after injury. SCI caused an increase of the ERK1/2 phosphorylation in vehicle-treated mice. The treatment with zileuton or montelukast reduced pERK1/2 levels. Densitometric analysis of protein expression represents the mean±s.e.mean of 10 spinal cord tissues. Data were normalized on the basis of ERK-2 levels. ° P

    Article Snippet: Rabbit monoclonal antibodies, anti-Bax, anti-bcl2 (Santa Cruz Biotechnology) and anti-COX-2 (Upstate cell signalling solutions, New York), and mouse monoclonal antibodies, anti-ERK-2 (Santa Cruz Biotechnology), were diluted 1:1000 in 0.1% PBS-Tween, 5% non-fat dry milk; mouse monoclonal antibody anti-pERK1/2 (Santa Cruz Biotechnology) was diluted 1:1000 in 0.1% PBS-Tween, 5% non-fat dry milk, 50 m M NaF; mouse monoclonal antibody anti-β-actin (Sigma-Aldrich, Milan, Italy) was diluted 1:10 000 in 0.1% PBS-Tween, 5% BSA.

    Techniques: Activation Assay, Western Blot, Mouse Assay, Expressing

    Effect of treatment with the raf inhibitor SB386023-b in cerebral arteries on the protein levels of pERK1/2 after SAH . There are increased expressions of the pERK1/2 protein levels in the SAH compared to the sham operated rats. Treatment with SB386023-b after 6 h prevented the increased protein expression. Data are presented as the pERK1/2/β-actin mean optical density ratio relative to control. Data are expressed as mean ± s.e.m. * P ≤ 0.05.

    Journal: BMC Neuroscience

    Article Title: Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

    doi: 10.1186/1471-2202-12-107

    Figure Lengend Snippet: Effect of treatment with the raf inhibitor SB386023-b in cerebral arteries on the protein levels of pERK1/2 after SAH . There are increased expressions of the pERK1/2 protein levels in the SAH compared to the sham operated rats. Treatment with SB386023-b after 6 h prevented the increased protein expression. Data are presented as the pERK1/2/β-actin mean optical density ratio relative to control. Data are expressed as mean ± s.e.m. * P ≤ 0.05.

    Article Snippet: Membranes were then incubated with the primary antibody of interest: pERK1/2 (1:5000 dilution; Promega, Madison, WI, U.S.A.) or β-actin (1:1000 dilution; Sigma, Saint Louis, USA) for 1 h at 37°C, followed by 3 × 5 min wash with T-TBS.

    Techniques: Expressing

    Sections from the basilar artery showing pERK immunoreactivity in the smooth muscle cell layer . a) pERK1/2; sham, b) pERK1/2; SAH, c) pERK; SAH treated with SB386023-b after 6 h, d) pERK; SAH treated with SB386023-b after 12 h, There are increased expressions of the pERK1/2 protein levels in the SAH compared to the sham operated rats. Treatment with SB386023-b after 6 h prevented the increased protein expression in the smooth muscle cells. Data were obtained with confocal microscopy.

    Journal: BMC Neuroscience

    Article Title: Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

    doi: 10.1186/1471-2202-12-107

    Figure Lengend Snippet: Sections from the basilar artery showing pERK immunoreactivity in the smooth muscle cell layer . a) pERK1/2; sham, b) pERK1/2; SAH, c) pERK; SAH treated with SB386023-b after 6 h, d) pERK; SAH treated with SB386023-b after 12 h, There are increased expressions of the pERK1/2 protein levels in the SAH compared to the sham operated rats. Treatment with SB386023-b after 6 h prevented the increased protein expression in the smooth muscle cells. Data were obtained with confocal microscopy.

    Article Snippet: Membranes were then incubated with the primary antibody of interest: pERK1/2 (1:5000 dilution; Promega, Madison, WI, U.S.A.) or β-actin (1:1000 dilution; Sigma, Saint Louis, USA) for 1 h at 37°C, followed by 3 × 5 min wash with T-TBS.

    Techniques: Expressing, Confocal Microscopy