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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
    Immunohistochemical analysis of phosphorylated ERK1/2 and total ERK2 in the striatum, hippocampus, and amygdala of WT, HT, and KO mice. Coronal brain sections from the three genotypes were double-labeled with antibodies that recognize the dually phosphorylated ERK1/2 (green) and total ERK2 (red). The two upper panels illustrate staining in the striatum (CPu) from the three genotypes. <t>pERK1/2</t> immunoreactivity was elevated in the KO mice when compared with WT and HT. The middle and bottom panels show representative sections from the hippocampus and amygdala, respectively. Increased pERK1/2 levels were noticed in the CA2 area of the hippocampus as well as central and lateral nuclei of the amygdala [Scale bar-50 μm].
    Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 3816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti perk1 2
    <t>pERK1/2+</t> cell counts. Following the adult behavior battery (chronic treatment with saline (SAL) or 122.2 mg/kg CaCl 2 in SAL (_Controls; equivalent amount of Ca 2 + ions as in the 300 mg/kg acamprosate treated group) or 300 mg/kg acamprosate in saline (+Acamp)), mice were sacrificed and brain sections were stained for pERK1/2 ( green ) and NeuN ( red ). As with the behavior measures, there were no differences in pERK1/ 2+ cell counts between the SAL- and CaCl 2 -treated mice and therefore data are presented as combined control groups (controls). In the dentate gyrus ( a , d – f ), there was a significant effect of drug with pairwise comparison testing demonstrating a trend towards an increase in pERK1/2 positive cells in the KO_Controls group (KO + SAL pictured in e ) compared to the untreated WT group (WT + SAL pictured in d ). Additionally, the KO + Acamp group ( f ) had significantly fewer pERK1/2+ cells than the KO + Controls. In the DG, all pERK1/2+ cells were also NeuN+. There were no differences in PERK1/2+ cell counts observed in the auditory cortex ( b ) or in the visual cortex ( c ). Data shown are LS mean ± SEM; * p
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    Santa Cruz Biotechnology perk1 2
    hucMSC-Ex Induces ERK1/2 Phosphorylation and Bcl2 Expression and Inhibits the IKKB/NFkB/Caspase-9/-3 Pathway (A) Western blot quantification of <t>pERK1/2,</t> total ERK1/2, Bcl2, pIKKβ, pNFkB, total NFkB, and casp9 and casp3. hucMSC-Ex induced ERK1/2 phosphorylation and Bcl2 expression and inhibited IKKB/NFkB/casp-9/-3 signaling in H 2 O 2 - or CCl 4 -injured L02 cells. (B) Bcl2 and pNFkB expression quantified by western blot (i). hucMSC-Ex dose-dependently inhibited pNFkB and induced Bcl2 expression in CCl 4 -injured L02 cells. Representative immunofluorescent images of pNFkB and Bcl2 expression in CCl 4 -injured L02 cells treated with PBS and hucMSC-Ex (ii). Original magnification 200×. (C) L02 cell viability was recovered by hucMSC-Ex or z-vad-FMK (n = 3). (D) CCl 4 -induced casp3 activity was reduced in hucMSC-Ex (20, 40, and 80 μg) (n = 3; *p
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    Cell Signaling Technology Inc rabbit anti perk1 2
    Expression of <t>pERK1/2</t> in intestinal mucosa at 4 and 7 days after MSCs transplantation. ( A ) Bands of the expression of pERK1/2 and its own tERk1/2 protein were detected by western blot. ( B ) The ratio value of pERK1/2 to its own tERK1/2 of the bands which was evaluated densitometrically using the software Quantity One for the three groups at different time points. Each bar represents mean ± SD of the ratio value in every group. S: sham group; C: control (saline) group; M: MSCs group. 4d and 7d represent 4 days and 7 days postoperatively. * : P
    Rabbit Anti Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 545 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
    Immunohistochemical staining results for <t>pERK1/2</t> (×10). Staining is performed on whisker from the Abcc6 knockout mouse (A-C) and PXE skin tissues (D-F) . Alizarin Red stains was used to localize mineralization (A and D) . Positive staining for pERK1/2 is obtained on murine whisker ( B, arrow) and PXE skin ( E, asterisk), co-localizing with mineralization. The pERK1/2 staining being broader than the mineralization staining could represent the active mineralization process where pERK1/2 expression precedes calcification. No staining is noted in control whisker (C) and control skin tissue (F) . (n = 5 each). Scale bar = 100 μm.
    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
    p38, JNK, and ERK1/2 activation regulated by NaO in S. aureus -challenged bMECs . MAPK phosphorylation was measured in bMECs that were treated with 0.25 or 1 mM NaO and/or challenged with S. aureus by flow cytometry. The phosphorylated MAPK concentrations (U/ml) are represented: (A) pp38, (B) pJNK1/2, and (C) <t>pERK1/2.</t> Each bar shows the result of one experiment. SB203580: p38 inhibitor. SP600125: JNK1/2 inhibitor. U0126: ERK1/2 inhibitor. Different letters above the bars indicate significant changes among the unchallenged bMECs (white bars) and S. aureus -challenged cells within the same MAPK evaluated ( 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
    Involvement of ERK1/2 <t>MAPK</t> pathway in Tat-mediated upregulation of K V 1.3 expression. A : Western blot analysis for ERK1/2 MAPK. Microglia were exposed to 200 ng/ml of Tat and harvested at indicated times. Protein expression was analyzed by immunoblot using antibodies against ERK1/2 phosphorylation <t>(pERK1/2)</t> and total ERK1/2 (ERK1/2) MAPK. Tat up-regulated pERK1/2 MAPK in a time window from 30 min to 5 hr. B : K v channel antagonists inhibit ERK1/2 MAPK phosphorylation. Microglia were treated with MgTx (5 nM), PAP (10 nM), or 4-AP (1 mM) for 30 min followed by Tat at 200 ng/ml for additional 5 hr. Gel blots reveal a reduction of Tat-induced ERK1/2 phosphorylation in microglia treated with MgTx, PAP, or 4-AP, indicating a link between K v 1.3 channel activation and ERK1/2 MAPK signal pathway. C : Western blot results showing that the blockade of Tat enhancement of K v 1.3 expression in microglia was blocked by U0126, an inhibitor for MEK1 and MEK 2, further demonstrating the link between ERK1/2 MAPK and Tat-induced increase of K v 1.3 expression. D : TUNEL staining exhibited a significant increase of neuronal apoptosis induced by the supernatants collected from Tat-treated microglia and its blockade by U0126, a MEK1 and MEK2 inhibitor. Data were from three independent experiments. *** p
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    Cell Signaling Technology Inc 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
    Perk1 2 Thr202 Tyr204, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse anti perk1 2
    ( a ) HCC827 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed by immuno-blotting using <t>anti-pERK1/2,</t> anti-pAkt, anti-pSrc and, anti-pEGFR (Tyr1068). Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, utilising Erk1/2, Akt, Src, EGFR and anti-tubulin antibodies. ( b ) H1975 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed as before. Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, as before
    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 anti perk1 2 t202 y204
    ( a ) HCC827 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed by immuno-blotting using <t>anti-pERK1/2,</t> anti-pAkt, anti-pSrc and, anti-pEGFR (Tyr1068). Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, utilising Erk1/2, Akt, Src, EGFR and anti-tubulin antibodies. ( b ) H1975 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed as before. Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, as before
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    Santa Cruz Biotechnology mouse anti perk1 2
    ( a ) HCC827 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed by immuno-blotting using <t>anti-pERK1/2,</t> anti-pAkt, anti-pSrc and, anti-pEGFR (Tyr1068). Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, utilising Erk1/2, Akt, Src, EGFR and anti-tubulin antibodies. ( b ) H1975 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed as before. Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, as before
    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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    Cell Signaling Technology Inc rabbit monoclonal anti perk1 2
    DEP-1 regulates FL-stimulated FLT3 signaling. DEP-1 expression was stably downregulated in THP-1 cells by lentiviral transduction of shRNA. Control cells harbor a non-targeting shRNA construct. ( A ) FL-dependent ERK1/2 activation was assessed by immunoblotting of cell lysate samples with antibodies recognizing activated ERK1/2 <t>(pERK1/2).</t> Loading was analyzed by reblot with ERK1/2 antibodies. Representative experiment; the efficiency of stable DEP-1 knockdown is also shown (upper panel). ( B ) Quantitative data for 4 independent experiments. Numbers represent pERK1/2 signals normalized to ERK1/2 levels in the same sample. The value of control cells at 10 min was set to 1.0. Significance for the difference between responses of the two different cell pools was determined by two-way ANOVA.
    Rabbit Monoclonal Anti Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit polyclonal anti perk1 2
    DEP-1 regulates FL-stimulated FLT3 signaling. DEP-1 expression was stably downregulated in THP-1 cells by lentiviral transduction of shRNA. Control cells harbor a non-targeting shRNA construct. ( A ) FL-dependent ERK1/2 activation was assessed by immunoblotting of cell lysate samples with antibodies recognizing activated ERK1/2 <t>(pERK1/2).</t> Loading was analyzed by reblot with ERK1/2 antibodies. Representative experiment; the efficiency of stable DEP-1 knockdown is also shown (upper panel). ( B ) Quantitative data for 4 independent experiments. Numbers represent pERK1/2 signals normalized to ERK1/2 levels in the same sample. The value of control cells at 10 min was set to 1.0. Significance for the difference between responses of the two different cell pools was determined by two-way ANOVA.
    Rabbit Polyclonal Anti Perk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 26 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
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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


    Immunohistochemical analysis of phosphorylated ERK1/2 and total ERK2 in the striatum, hippocampus, and amygdala of WT, HT, and KO mice. Coronal brain sections from the three genotypes were double-labeled with antibodies that recognize the dually phosphorylated ERK1/2 (green) and total ERK2 (red). The two upper panels illustrate staining in the striatum (CPu) from the three genotypes. pERK1/2 immunoreactivity was elevated in the KO mice when compared with WT and HT. The middle and bottom panels show representative sections from the hippocampus and amygdala, respectively. Increased pERK1/2 levels were noticed in the CA2 area of the hippocampus as well as central and lateral nuclei of the amygdala [Scale bar-50 μm].

    Journal: Synapse (New York, N.Y.)

    Article Title: Knockout of STriatal Enriched Protein Tyrosine Phosphatase in Mice Results in Increased ERK1/2 Phosphorylation

    doi: 10.1002/syn.20608

    Figure Lengend Snippet: Immunohistochemical analysis of phosphorylated ERK1/2 and total ERK2 in the striatum, hippocampus, and amygdala of WT, HT, and KO mice. Coronal brain sections from the three genotypes were double-labeled with antibodies that recognize the dually phosphorylated ERK1/2 (green) and total ERK2 (red). The two upper panels illustrate staining in the striatum (CPu) from the three genotypes. pERK1/2 immunoreactivity was elevated in the KO mice when compared with WT and HT. The middle and bottom panels show representative sections from the hippocampus and amygdala, respectively. Increased pERK1/2 levels were noticed in the CA2 area of the hippocampus as well as central and lateral nuclei of the amygdala [Scale bar-50 μm].

    Article Snippet: The levels of pERK1/2 were significantly elevated in both subcellular fractions obtained from the striatum of the KO mice (S2: 179% ± 14%; P < 0.03 and P2: 169% ± 12%; P < 0.001) ( ).

    Techniques: Immunohistochemistry, Mouse Assay, Labeling, Staining

    Enhanced phosphorylation of ERK1/2 in the KO hippocampal cultures and synaptoneurosomes. ( A ) The STEP WT and KO cultures were stimulated with increasing concentration of DHPG for 5 min and stained with pERK1/2 antibody. DHPG leads to activation of pERK1/2 in both the WT and KO cultures. However, the phosphorylation of ERK1/2 is more pronounced in the KO cells when compared with WT. This suggests that in the absence of STEP, there is a higher level of pERK1/2 activation [Scale bar-50 μm]. ( B ) Synaptoneurosomes prepared from WT and KO mice hippocampi were stimulated with increasing concentration of DHPG and processed for Western blotting. The membranes were probed with pERK1/2 and ERK2 antibodies sequentially. ( C ) The pERK1/2 levels from WT and KO synaptoneurosomes were measured using ImageJ and normalized to total ERK2 levels. The normalized pERK1/2 levels are plotted as a percentage of WT control levels. The data were compared using one-way ANOVA, followed by posthoc Tukey HSD (WT 20 μm: 126% ± 2%, P > 0.1; WT 50 μm: 138% ± 7%, P

    Journal: Synapse (New York, N.Y.)

    Article Title: Knockout of STriatal Enriched Protein Tyrosine Phosphatase in Mice Results in Increased ERK1/2 Phosphorylation

    doi: 10.1002/syn.20608

    Figure Lengend Snippet: Enhanced phosphorylation of ERK1/2 in the KO hippocampal cultures and synaptoneurosomes. ( A ) The STEP WT and KO cultures were stimulated with increasing concentration of DHPG for 5 min and stained with pERK1/2 antibody. DHPG leads to activation of pERK1/2 in both the WT and KO cultures. However, the phosphorylation of ERK1/2 is more pronounced in the KO cells when compared with WT. This suggests that in the absence of STEP, there is a higher level of pERK1/2 activation [Scale bar-50 μm]. ( B ) Synaptoneurosomes prepared from WT and KO mice hippocampi were stimulated with increasing concentration of DHPG and processed for Western blotting. The membranes were probed with pERK1/2 and ERK2 antibodies sequentially. ( C ) The pERK1/2 levels from WT and KO synaptoneurosomes were measured using ImageJ and normalized to total ERK2 levels. The normalized pERK1/2 levels are plotted as a percentage of WT control levels. The data were compared using one-way ANOVA, followed by posthoc Tukey HSD (WT 20 μm: 126% ± 2%, P > 0.1; WT 50 μm: 138% ± 7%, P

    Article Snippet: The levels of pERK1/2 were significantly elevated in both subcellular fractions obtained from the striatum of the KO mice (S2: 179% ± 14%; P < 0.03 and P2: 169% ± 12%; P < 0.001) ( ).

    Techniques: Concentration Assay, Staining, Activation Assay, Mouse Assay, Western Blot

    pERK1/2+ cell counts. Following the adult behavior battery (chronic treatment with saline (SAL) or 122.2 mg/kg CaCl 2 in SAL (_Controls; equivalent amount of Ca 2 + ions as in the 300 mg/kg acamprosate treated group) or 300 mg/kg acamprosate in saline (+Acamp)), mice were sacrificed and brain sections were stained for pERK1/2 ( green ) and NeuN ( red ). As with the behavior measures, there were no differences in pERK1/ 2+ cell counts between the SAL- and CaCl 2 -treated mice and therefore data are presented as combined control groups (controls). In the dentate gyrus ( a , d – f ), there was a significant effect of drug with pairwise comparison testing demonstrating a trend towards an increase in pERK1/2 positive cells in the KO_Controls group (KO + SAL pictured in e ) compared to the untreated WT group (WT + SAL pictured in d ). Additionally, the KO + Acamp group ( f ) had significantly fewer pERK1/2+ cells than the KO + Controls. In the DG, all pERK1/2+ cells were also NeuN+. There were no differences in PERK1/2+ cell counts observed in the auditory cortex ( b ) or in the visual cortex ( c ). Data shown are LS mean ± SEM; * p

    Journal: Journal of Neurodevelopmental Disorders

    Article Title: Acamprosate in a mouse model of fragile X syndrome: modulation of spontaneous cortical activity, ERK1/2 activation, locomotor behavior, and anxiety

    doi: 10.1186/s11689-017-9184-y

    Figure Lengend Snippet: pERK1/2+ cell counts. Following the adult behavior battery (chronic treatment with saline (SAL) or 122.2 mg/kg CaCl 2 in SAL (_Controls; equivalent amount of Ca 2 + ions as in the 300 mg/kg acamprosate treated group) or 300 mg/kg acamprosate in saline (+Acamp)), mice were sacrificed and brain sections were stained for pERK1/2 ( green ) and NeuN ( red ). As with the behavior measures, there were no differences in pERK1/ 2+ cell counts between the SAL- and CaCl 2 -treated mice and therefore data are presented as combined control groups (controls). In the dentate gyrus ( a , d – f ), there was a significant effect of drug with pairwise comparison testing demonstrating a trend towards an increase in pERK1/2 positive cells in the KO_Controls group (KO + SAL pictured in e ) compared to the untreated WT group (WT + SAL pictured in d ). Additionally, the KO + Acamp group ( f ) had significantly fewer pERK1/2+ cells than the KO + Controls. In the DG, all pERK1/2+ cells were also NeuN+. There were no differences in PERK1/2+ cell counts observed in the auditory cortex ( b ) or in the visual cortex ( c ). Data shown are LS mean ± SEM; * p

    Article Snippet: Sections were then blocked in 10% normal donkey serum (NDS) for 1 h. Sections were incubated in 1:400 rabbit, anti-pERK1/2 primary antibody (#4370; Cell Signaling) for 48 h followed by incubation in 1:200 swine, anti-rabbit, biotinylated secondary antibody (E0353; Dako) solution for 3 h. Following secondary, tissue was incubated for 1 h in ABC solution (VECTASTAIN Elite ABC HRP Kit; Vector) which was prepared 30 min prior to use.

    Techniques: Mouse Assay, Staining

    Increased expression of SA-pErk1/2 and transcription of p21 Sdi1 by active PKCα. A , pcDNA3-HA ( vector ), dominant negative PKC-α (PKCα-DN), or catalytically active PKC-α ( PKC α- CA ) were introduced into HDF young cells by electroporation, and the whole cell lysates (30 μg) were subjected to immunoblot analyses in 2 days. Note significant inductions of SA-pErk1/2 and p21 Sdi1 by PKCα-CA, as opposed to down-regulation of pErk1/2 expression by PKCα-DN. B , treatment of HDF young cells with DiC 8 (20 μg/ml), a PKC activator, for 7 days also increased p21 Sdi1 and SA-pErk1/2 expressions, and concomitantly decreased cell growth ( C ). To prove regulations of the above mentioned phenotypes by PKCα activity, p21 Sdi1 promoter-driven luciferase assay was performed ( D ); NIH3T3 cells were co-transfected with the reporter plasmid, p21 Sdi1 promoter-luciferase (pGL2-0.3), along with pcDNA3-HA (vector), PKCα-DN, or PKCα-CA for 48 h. The cells were homogenized and then subjected to luciferase ( Luc ) assay. Note the significant activity of the p21 Sdi1 transcription by PKCα-CA, but not PKCα-DN. All of the data indicate mean ± S.D. from three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1 *

    doi: 10.1074/jbc.M808734200

    Figure Lengend Snippet: Increased expression of SA-pErk1/2 and transcription of p21 Sdi1 by active PKCα. A , pcDNA3-HA ( vector ), dominant negative PKC-α (PKCα-DN), or catalytically active PKC-α ( PKC α- CA ) were introduced into HDF young cells by electroporation, and the whole cell lysates (30 μg) were subjected to immunoblot analyses in 2 days. Note significant inductions of SA-pErk1/2 and p21 Sdi1 by PKCα-CA, as opposed to down-regulation of pErk1/2 expression by PKCα-DN. B , treatment of HDF young cells with DiC 8 (20 μg/ml), a PKC activator, for 7 days also increased p21 Sdi1 and SA-pErk1/2 expressions, and concomitantly decreased cell growth ( C ). To prove regulations of the above mentioned phenotypes by PKCα activity, p21 Sdi1 promoter-driven luciferase assay was performed ( D ); NIH3T3 cells were co-transfected with the reporter plasmid, p21 Sdi1 promoter-luciferase (pGL2-0.3), along with pcDNA3-HA (vector), PKCα-DN, or PKCα-CA for 48 h. The cells were homogenized and then subjected to luciferase ( Luc ) assay. Note the significant activity of the p21 Sdi1 transcription by PKCα-CA, but not PKCα-DN. All of the data indicate mean ± S.D. from three independent experiments.

    Article Snippet: Anti-pErk1/2 antibody was from Cell Signaling; antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCϵ, PKCη, PKCζ, Sp1, and p21Sdi1 were from Santa Cruz; anti-p53 and α-tubulin antibodies were from Oncogene.

    Techniques: Expressing, Plasmid Preparation, Dominant Negative Mutation, Electroporation, Activity Assay, Luciferase, Transfection

    In vitro phosphorylation of GST-Sp1 by PKCα and Erk1. To investigate whether phosphorylation of Sp1 was regulated directly by PKCα or SA-pErk1/2, a human Sp1 fusion construct ( GST-Sp1 ) and its deletion mutants ( GST -Δ 1 , 1–110 residues of Sp1; GST -Δ 2 , 301–350 residues of Sp1; GST -Δ 3 , 504–785 residues of Sp1) were prepared as described under “Experimental Procedures,” focusing on the potential phosphorylation sites ( A ). The recombinant proteins were expressed in E. coli and confirmed by Coomassie Blue stain after SDS-PAGE ( B ). In vitro phosphorylations of Sp1 by PKCα ( C ) and active Erk1 ( D ) were evaluated by an in vitro kinase assay. The recombinant proteins were subjected to kinase assay in the presence of 5 μCi of [γ- 32 P]ATP and 10 times excess amount of unlabeled ATP. The reaction mixture was separated by SDS-PAGE and then visualized by autoradiography. Note phosphorylated GST-Δ1 and GST-Δ3, but not GST-Δ2, in addition to the GST-Sp1 full sequence by active PKCα and active Erk1. To identify phosphorylated residues in the recombinant proteins, immunoblot ( IB ) analyses were performed with anti-Ser(P) ( E ) and anti-Thr(P) ( F ) antibodies. Note the phosphorylations of serine and threonine residues in the GST-Δ1 and GST-Δ3, respectively, by active Erk1.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1 *

    doi: 10.1074/jbc.M808734200

    Figure Lengend Snippet: In vitro phosphorylation of GST-Sp1 by PKCα and Erk1. To investigate whether phosphorylation of Sp1 was regulated directly by PKCα or SA-pErk1/2, a human Sp1 fusion construct ( GST-Sp1 ) and its deletion mutants ( GST -Δ 1 , 1–110 residues of Sp1; GST -Δ 2 , 301–350 residues of Sp1; GST -Δ 3 , 504–785 residues of Sp1) were prepared as described under “Experimental Procedures,” focusing on the potential phosphorylation sites ( A ). The recombinant proteins were expressed in E. coli and confirmed by Coomassie Blue stain after SDS-PAGE ( B ). In vitro phosphorylations of Sp1 by PKCα ( C ) and active Erk1 ( D ) were evaluated by an in vitro kinase assay. The recombinant proteins were subjected to kinase assay in the presence of 5 μCi of [γ- 32 P]ATP and 10 times excess amount of unlabeled ATP. The reaction mixture was separated by SDS-PAGE and then visualized by autoradiography. Note phosphorylated GST-Δ1 and GST-Δ3, but not GST-Δ2, in addition to the GST-Sp1 full sequence by active PKCα and active Erk1. To identify phosphorylated residues in the recombinant proteins, immunoblot ( IB ) analyses were performed with anti-Ser(P) ( E ) and anti-Thr(P) ( F ) antibodies. Note the phosphorylations of serine and threonine residues in the GST-Δ1 and GST-Δ3, respectively, by active Erk1.

    Article Snippet: Anti-pErk1/2 antibody was from Cell Signaling; antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCϵ, PKCη, PKCζ, Sp1, and p21Sdi1 were from Santa Cruz; anti-p53 and α-tubulin antibodies were from Oncogene.

    Techniques: In Vitro, Construct, Recombinant, Staining, SDS Page, Kinase Assay, Autoradiography, Sequencing

    Phosphorylation of Sp1 on Ser 59 by SA-pErk1/2 downstream of PKCα, and induction of p21 Sdi1 expression. ROS and PKCα concurrently stimulated each other during cellular senescence. Accumulated ROS stimulates the activity of PKCα, and the active PKCα in turn stimulates ROS generation during cellular senescence, demonstrated by treatment of the old cells with PKCα-siRNA. Moreover, activated PKCα regulates the expression of senescence-associated pErk1/2 in the cytoplasm ( SA-pErk1 / 2 ), which in turn increases transcription of p21 Sdi1 via in vivo phosphorylation of Sp1 on Ser 59 . Elevated p21 Sdi1 induces cell-cycle arrest of the actively growing cells at G 1 phase, leading to growth arrest of senescent cells. On the other hand, down-regulation of PKCα expression by treatment of old cells with PKCα-siRNA significantly reduces SA-pErk1/2 in both nuclear and cytoplasmic fractions of the old cells, and inhibits Sp1 phosphorylation on Ser 59 , but not Thr 739 , and transcription of p21 Sdi1 , resulting in the release of senescent cells from G 1 arrest. In summary, in vivo regulation of p21 Sdi1 expression occurs via Sp1 phosphorylation on Ser 59 by pErk1/2 downstream of PKCα in the process of cellular senescence.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1 *

    doi: 10.1074/jbc.M808734200

    Figure Lengend Snippet: Phosphorylation of Sp1 on Ser 59 by SA-pErk1/2 downstream of PKCα, and induction of p21 Sdi1 expression. ROS and PKCα concurrently stimulated each other during cellular senescence. Accumulated ROS stimulates the activity of PKCα, and the active PKCα in turn stimulates ROS generation during cellular senescence, demonstrated by treatment of the old cells with PKCα-siRNA. Moreover, activated PKCα regulates the expression of senescence-associated pErk1/2 in the cytoplasm ( SA-pErk1 / 2 ), which in turn increases transcription of p21 Sdi1 via in vivo phosphorylation of Sp1 on Ser 59 . Elevated p21 Sdi1 induces cell-cycle arrest of the actively growing cells at G 1 phase, leading to growth arrest of senescent cells. On the other hand, down-regulation of PKCα expression by treatment of old cells with PKCα-siRNA significantly reduces SA-pErk1/2 in both nuclear and cytoplasmic fractions of the old cells, and inhibits Sp1 phosphorylation on Ser 59 , but not Thr 739 , and transcription of p21 Sdi1 , resulting in the release of senescent cells from G 1 arrest. In summary, in vivo regulation of p21 Sdi1 expression occurs via Sp1 phosphorylation on Ser 59 by pErk1/2 downstream of PKCα in the process of cellular senescence.

    Article Snippet: Anti-pErk1/2 antibody was from Cell Signaling; antibodies against PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCϵ, PKCη, PKCζ, Sp1, and p21Sdi1 were from Santa Cruz; anti-p53 and α-tubulin antibodies were from Oncogene.

    Techniques: Expressing, Activity Assay, In Vivo

    Effects of PD98059, a pERK1/2 inhibitor, on LPS-mediated COX-2 expression in AGS cells. ( a ) Cells were treated with and without 10 ng/mL LPS for 15, 30, and 60 min. Cell lysates were harvested for a pERK1/2 expression analysis. To further determine the effect of PD98059 on LPS-medicated COX-2 , cells were transiently transfected with 0.5 μg of ( b ) a COX-2 promoter reporter construct (pXC918) and ( c ) 3X NF-κB reporter construct for 24 h. Cells were incubated with 10 μM PD98059 for 30 min, and stimulated with 10 ng/mL LPS for 2 h. Promoter activity was measured with a luciferase assay. Statistical significance (** p

    Journal: Scientific Reports

    Article Title: Store-operated Ca2+ Entry Facilitates the Lipopolysaccharide-induced Cyclooxygenase-2 Expression in Gastric Cancer Cells

    doi: 10.1038/s41598-017-12648-1

    Figure Lengend Snippet: Effects of PD98059, a pERK1/2 inhibitor, on LPS-mediated COX-2 expression in AGS cells. ( a ) Cells were treated with and without 10 ng/mL LPS for 15, 30, and 60 min. Cell lysates were harvested for a pERK1/2 expression analysis. To further determine the effect of PD98059 on LPS-medicated COX-2 , cells were transiently transfected with 0.5 μg of ( b ) a COX-2 promoter reporter construct (pXC918) and ( c ) 3X NF-κB reporter construct for 24 h. Cells were incubated with 10 μM PD98059 for 30 min, and stimulated with 10 ng/mL LPS for 2 h. Promoter activity was measured with a luciferase assay. Statistical significance (** p

    Article Snippet: Antibodies against pERK1/2 (Cell Signaling, Beverly, MA), STIM1 (Cell Signaling, Beverly, MA), and ORAI1 (GeneTex, Hsinchu, Taiwan) were diluted 1: 2000, whereas the antibody against β-actin was diluted 1: 10000.

    Techniques: Expressing, Transfection, Construct, Incubation, Activity Assay, Luciferase

    Western blot of total ERK1/2 ( 4a ) and pERK1/2 ( 4b ) in untreated, hCG treated and LH treated cultures at different time points. hCG and LH treated cultures show similar expression whereas there is no expression of pERK1/2 in untreated cultures at 0 and 40 min (L-ladder, C-positive control). Anti-β actin antibody was used to ensure equal loading.

    Journal: The Indian Journal of Medical Research

    Article Title: Differential response to sustained stimulation by hCG LH on goat ovarian granulosa cells

    doi:

    Figure Lengend Snippet: Western blot of total ERK1/2 ( 4a ) and pERK1/2 ( 4b ) in untreated, hCG treated and LH treated cultures at different time points. hCG and LH treated cultures show similar expression whereas there is no expression of pERK1/2 in untreated cultures at 0 and 40 min (L-ladder, C-positive control). Anti-β actin antibody was used to ensure equal loading.

    Article Snippet: Total ERK1/2 antibody and pERK1/2 antibody were procured from Cell Signaling Technology, USA.

    Techniques: Western Blot, Expressing, Positive Control

    Incubation with recombinant Wnt5a, but not recombinant Wnt3a nor EGF enhanced villin protein expression . (A) In WT Ror2-transfected HT29 cells, villin protein was slightly increased compared to Wnt5a CM-treated mock-transfected cells. There was a noticeable increase in similarly treated WT Ror2-overexpressing cells. Incubation with rWnt5a (200 ng/mL) for 24 h enhanced villin expression to a similar degree. Treatment with rWnt3a (200 ng/mL) for 24 h did not induce any immunoreactive villin expression. (B) Challenge with EGF (100 nM) for 0, 2, 5, 10, 30, 60 min induced transient and robust increases in ERK1/2 phosphorylation in HT29 cells. In contrast, weak pERK1/2 immunoreactivity was detected in cells which had been challenged with low serum media, which did not contain EGF, over similar time points. (C) As previously observed, in HT29 cells which were mock-transfected or left untreated, low levels of villin protein expression were detected. In WT Ror2-overexpressing cells challenged with Wnt5a conditioned media for 24 h, villin protein expression was increased. In WT Ror2-overexpressing cells, challenged with EGF for a similar 24 h time period, low levels of villin expression were detectable, comparable to that found in mock-transfected or untreated control cells. β-actin immunoblotting was included as a loading control. These results are representative of three separately performed experiments.

    Journal: Frontiers in Physiology

    Article Title: Regulation of Villin by Wnt5a/Ror2 Signaling in Human Intestinal Cells

    doi: 10.3389/fphys.2011.00058

    Figure Lengend Snippet: Incubation with recombinant Wnt5a, but not recombinant Wnt3a nor EGF enhanced villin protein expression . (A) In WT Ror2-transfected HT29 cells, villin protein was slightly increased compared to Wnt5a CM-treated mock-transfected cells. There was a noticeable increase in similarly treated WT Ror2-overexpressing cells. Incubation with rWnt5a (200 ng/mL) for 24 h enhanced villin expression to a similar degree. Treatment with rWnt3a (200 ng/mL) for 24 h did not induce any immunoreactive villin expression. (B) Challenge with EGF (100 nM) for 0, 2, 5, 10, 30, 60 min induced transient and robust increases in ERK1/2 phosphorylation in HT29 cells. In contrast, weak pERK1/2 immunoreactivity was detected in cells which had been challenged with low serum media, which did not contain EGF, over similar time points. (C) As previously observed, in HT29 cells which were mock-transfected or left untreated, low levels of villin protein expression were detected. In WT Ror2-overexpressing cells challenged with Wnt5a conditioned media for 24 h, villin protein expression was increased. In WT Ror2-overexpressing cells, challenged with EGF for a similar 24 h time period, low levels of villin expression were detectable, comparable to that found in mock-transfected or untreated control cells. β-actin immunoblotting was included as a loading control. These results are representative of three separately performed experiments.

    Article Snippet: To detect expression of phosphorylated-ERK1/2 (pERK1/2), total amounts of ERK1/2 (ERK1/2), Ror2, FLAG-tag, and villin protein, membranes were incubated with a polyclonal rabbit antibody specific for pERK1/2, ERK1/2, Ror2, FLAG-tag, or villin in 1XTBS-T with 5% BSA in a 1:1000 dilution (antibodies purchased from Cell Signaling Technology Inc., Danvers, MA, USA).

    Techniques: Incubation, Recombinant, Expressing, Transfection

    hucMSC-Ex Induces ERK1/2 Phosphorylation and Bcl2 Expression and Inhibits the IKKB/NFkB/Caspase-9/-3 Pathway (A) Western blot quantification of pERK1/2, total ERK1/2, Bcl2, pIKKβ, pNFkB, total NFkB, and casp9 and casp3. hucMSC-Ex induced ERK1/2 phosphorylation and Bcl2 expression and inhibited IKKB/NFkB/casp-9/-3 signaling in H 2 O 2 - or CCl 4 -injured L02 cells. (B) Bcl2 and pNFkB expression quantified by western blot (i). hucMSC-Ex dose-dependently inhibited pNFkB and induced Bcl2 expression in CCl 4 -injured L02 cells. Representative immunofluorescent images of pNFkB and Bcl2 expression in CCl 4 -injured L02 cells treated with PBS and hucMSC-Ex (ii). Original magnification 200×. (C) L02 cell viability was recovered by hucMSC-Ex or z-vad-FMK (n = 3). (D) CCl 4 -induced casp3 activity was reduced in hucMSC-Ex (20, 40, and 80 μg) (n = 3; *p

    Journal: Molecular Therapy

    Article Title: hucMSC Exosome-Derived GPX1 Is Required for the Recovery of Hepatic Oxidant Injury

    doi: 10.1016/j.ymthe.2016.11.019

    Figure Lengend Snippet: hucMSC-Ex Induces ERK1/2 Phosphorylation and Bcl2 Expression and Inhibits the IKKB/NFkB/Caspase-9/-3 Pathway (A) Western blot quantification of pERK1/2, total ERK1/2, Bcl2, pIKKβ, pNFkB, total NFkB, and casp9 and casp3. hucMSC-Ex induced ERK1/2 phosphorylation and Bcl2 expression and inhibited IKKB/NFkB/casp-9/-3 signaling in H 2 O 2 - or CCl 4 -injured L02 cells. (B) Bcl2 and pNFkB expression quantified by western blot (i). hucMSC-Ex dose-dependently inhibited pNFkB and induced Bcl2 expression in CCl 4 -injured L02 cells. Representative immunofluorescent images of pNFkB and Bcl2 expression in CCl 4 -injured L02 cells treated with PBS and hucMSC-Ex (ii). Original magnification 200×. (C) L02 cell viability was recovered by hucMSC-Ex or z-vad-FMK (n = 3). (D) CCl 4 -induced casp3 activity was reduced in hucMSC-Ex (20, 40, and 80 μg) (n = 3; *p

    Article Snippet: Transferred membranes were incubated with the primary antibodies against Bcl2 (Bioworld), CD9 (Bioworld), CD61 (Bioworld), CD63 (Bioworld), pERK1/2 (Santa Cruz), ERK1/2 (Santa Cruz), pIKKβ (SAB), pNFkB (SAB), NFkB (SAB), Casp9 (Santa Cruz), Casp3 (Santa Cruz), cleaved Casp3 (Santa Cruz), and GAPDH (KangCheng) overnight at 4°C.

    Techniques: Expressing, Western Blot, Activity Assay

    Effect of shGnb2l1 icv injection on ERK/CREB pathway. ( a ) shGnb2l1 icv injection significantly decreased the expression of pERK1/2 protein in the CA2 of HP in acquisition and maintenance. The brown cells represent the pERK1/2 positive cells. The scale bar = 0.5 mm. ( a,f ) saline + shNC mouse; ( b,g ) saline + shGnb2l1 mouse; ( c,h ) morphine + shNC mouse; ( d,i ) morphine + shGnb2l1 mouse; ( e ) Enlarged, from ( h ) shows that pyramidal neurons (Pyr) in the DG of the HP are strongly positive for pERK1/2; ( a–d,f–i ) The scale bar = 2 mm; ( e ) The scale bar = 0.5 mm.( b ) shGnb2l1 icv injection significantly decreased the expression of pCREB protein in the CA1 of HP in acquisition and maintenance. The brown cells represent the pCREB positive cells in the DAB immunostaining. The scale bar = 0.5 mm. ( a,f ) saline + shNC mouse; ( b,g ) saline + shGnb2l1 mouse; ( c,h ) morphine + shNC mouse; ( d,i ) morphine + shGnb2l1 mouse; ( e ) Enlarged, from ( c ) shows that pyramidal neurons (Pyr) in the CA1of the HP are strongly positive for pCREB; ( a–d,f–i ) The scale bar = 2 mm; ( e ) The scale bar = 0.5 mm. ( c ) Representative immunoblot showing the molecular weights of the proteins used to quantify level of ERK1/2 (44/42 kDa), pERK1/2 (43 kDa), CREB (45 kDa) and pCREB (43 kDa) in HP. Signals were normalized to β-actin (43 kDa). The level of ERK1/2 and CREB in HP were not changed after morphine treatment in acquisition and maintenance experiment. An increase in pERK1/2 and pCREB in HP could be detected in both experiments. shGnb2l1icv injection significantly decreased the protein expressions of pERK1/2 and pCREB in HP; ( d ) Comparison of the ratio of the pERK/ERK band between the experimental groups, * p

    Journal: Scientific Reports

    Article Title: RACK1 promotes maintenance of morphine-associated memory via activation of an ERK-CREB dependent pathway in hippocampus

    doi: 10.1038/srep20183

    Figure Lengend Snippet: Effect of shGnb2l1 icv injection on ERK/CREB pathway. ( a ) shGnb2l1 icv injection significantly decreased the expression of pERK1/2 protein in the CA2 of HP in acquisition and maintenance. The brown cells represent the pERK1/2 positive cells. The scale bar = 0.5 mm. ( a,f ) saline + shNC mouse; ( b,g ) saline + shGnb2l1 mouse; ( c,h ) morphine + shNC mouse; ( d,i ) morphine + shGnb2l1 mouse; ( e ) Enlarged, from ( h ) shows that pyramidal neurons (Pyr) in the DG of the HP are strongly positive for pERK1/2; ( a–d,f–i ) The scale bar = 2 mm; ( e ) The scale bar = 0.5 mm.( b ) shGnb2l1 icv injection significantly decreased the expression of pCREB protein in the CA1 of HP in acquisition and maintenance. The brown cells represent the pCREB positive cells in the DAB immunostaining. The scale bar = 0.5 mm. ( a,f ) saline + shNC mouse; ( b,g ) saline + shGnb2l1 mouse; ( c,h ) morphine + shNC mouse; ( d,i ) morphine + shGnb2l1 mouse; ( e ) Enlarged, from ( c ) shows that pyramidal neurons (Pyr) in the CA1of the HP are strongly positive for pCREB; ( a–d,f–i ) The scale bar = 2 mm; ( e ) The scale bar = 0.5 mm. ( c ) Representative immunoblot showing the molecular weights of the proteins used to quantify level of ERK1/2 (44/42 kDa), pERK1/2 (43 kDa), CREB (45 kDa) and pCREB (43 kDa) in HP. Signals were normalized to β-actin (43 kDa). The level of ERK1/2 and CREB in HP were not changed after morphine treatment in acquisition and maintenance experiment. An increase in pERK1/2 and pCREB in HP could be detected in both experiments. shGnb2l1icv injection significantly decreased the protein expressions of pERK1/2 and pCREB in HP; ( d ) Comparison of the ratio of the pERK/ERK band between the experimental groups, * p

    Article Snippet: Subsequently, the sections were incubated overnight with a goat polyclonal anti-RACK1 antibody (Santa Cruz Biotechnology, Inc., USA, 1:100), anti-pCREB (Santa Cruz Biotechnology, Inc., USA, 1:100), anti-pERK1/2 (Santa Cruz Biotechnology, Inc., USA, 1:100), and anti-SYP (Abcam, Cambridge, UK, 1:100), antibody at 4 °C, followed by the addition of biotinylated rabbit anti-goat IgG secondary antibody (Jinshan, BJ, China).

    Techniques: Injection, Expressing, Immunostaining

    Expression of pERK1/2 in intestinal mucosa at 4 and 7 days after MSCs transplantation. ( A ) Bands of the expression of pERK1/2 and its own tERk1/2 protein were detected by western blot. ( B ) The ratio value of pERK1/2 to its own tERK1/2 of the bands which was evaluated densitometrically using the software Quantity One for the three groups at different time points. Each bar represents mean ± SD of the ratio value in every group. S: sham group; C: control (saline) group; M: MSCs group. 4d and 7d represent 4 days and 7 days postoperatively. * : P

    Journal: PLoS ONE

    Article Title: Potential Role of Mesenchymal Stem Cells in Alleviating Intestinal Ischemia/Reperfusion Impairment

    doi: 10.1371/journal.pone.0074468

    Figure Lengend Snippet: Expression of pERK1/2 in intestinal mucosa at 4 and 7 days after MSCs transplantation. ( A ) Bands of the expression of pERK1/2 and its own tERk1/2 protein were detected by western blot. ( B ) The ratio value of pERK1/2 to its own tERK1/2 of the bands which was evaluated densitometrically using the software Quantity One for the three groups at different time points. Each bar represents mean ± SD of the ratio value in every group. S: sham group; C: control (saline) group; M: MSCs group. 4d and 7d represent 4 days and 7 days postoperatively. * : P

    Article Snippet: The membranes were blocked in blocking solution (0.1% Triton-X-100, 15 mmol/L NaCl, 2 mmol/L Tris-HCl, pH 7.5) containing 3% bovine serum albumin (BSA) for 30mins, then incubated overnight with rabbit anti-pERK1/2 monoclonal antibody (Cell Signaling, US, 1:100), rabbit anti-lamin B1 polyclonal antibody (Abcam, UK; 1:500), and rabbit anti-NF-kB p65 antibody (Santa Cruz, US; 1:100) diluted in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 1% BSA at 4°C, then washed three times (5 mins each time) with TBST.

    Techniques: Expressing, Transplantation Assay, Western Blot, Software

    The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class III mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Journal: International Journal of Biological Sciences

    Article Title: Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

    doi: 10.7150/ijbs.10359

    Figure Lengend Snippet: The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class III mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Article Snippet: Phospho-ERK1/2 and β-tubulin were detected by immunoblotting with 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 the University of Iowa, Iowa City, IA), respectively.

    Techniques: Transfection

    The basal pERK1/2 levels in HEK293T cells transiently transfected with WT or mutant MC4Rs with increased basal pERK1/2 levels (A), or decreased basal pERK1/2 levels (B and C). Results are expressed as percentage of the WT basal pERK1/2 level and represent the mean ± SEM of five independent experiments. * indicates significant differences (P

    Journal: International Journal of Biological Sciences

    Article Title: Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

    doi: 10.7150/ijbs.10359

    Figure Lengend Snippet: The basal pERK1/2 levels in HEK293T cells transiently transfected with WT or mutant MC4Rs with increased basal pERK1/2 levels (A), or decreased basal pERK1/2 levels (B and C). Results are expressed as percentage of the WT basal pERK1/2 level and represent the mean ± SEM of five independent experiments. * indicates significant differences (P

    Article Snippet: Phospho-ERK1/2 and β-tubulin were detected by immunoblotting with 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 the University of Iowa, Iowa City, IA), respectively.

    Techniques: Transfection, Mutagenesis

    The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class I mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Journal: International Journal of Biological Sciences

    Article Title: Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

    doi: 10.7150/ijbs.10359

    Figure Lengend Snippet: The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class I mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Article Snippet: Phospho-ERK1/2 and β-tubulin were detected by immunoblotting with 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 the University of Iowa, Iowa City, IA), respectively.

    Techniques: Transfection

    The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class V variants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant difference from basal pERK1/2 level or from the stimulation of the WT MC4R (P

    Journal: International Journal of Biological Sciences

    Article Title: Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

    doi: 10.7150/ijbs.10359

    Figure Lengend Snippet: The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class V variants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant difference from basal pERK1/2 level or from the stimulation of the WT MC4R (P

    Article Snippet: Phospho-ERK1/2 and β-tubulin were detected by immunoblotting with 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 the University of Iowa, Iowa City, IA), respectively.

    Techniques: Transfection

    The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class IV mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Journal: International Journal of Biological Sciences

    Article Title: Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

    doi: 10.7150/ijbs.10359

    Figure Lengend Snippet: The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class IV mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Article Snippet: Phospho-ERK1/2 and β-tubulin were detected by immunoblotting with 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 the University of Iowa, Iowa City, IA), respectively.

    Techniques: Transfection

    The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class II mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Journal: International Journal of Biological Sciences

    Article Title: Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

    doi: 10.7150/ijbs.10359

    Figure Lengend Snippet: The ligand-stimulated pERK1/2 levels in HEK293T cells transiently transfected with Class II mutants. Results are expressed as percentage of the value obtained in non-stimulated cells and represent the mean ± SEM of five independent experiments. * indicates significant differences from basal pERK1/2 level (P

    Article Snippet: Phospho-ERK1/2 and β-tubulin were detected by immunoblotting with 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 the University of Iowa, Iowa City, IA), respectively.

    Techniques: Transfection

    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: Antibodies against ARAF (#4432), pBRAF S445 (#2696), pCRAF S338 (#9427), pEGFR Y1068 (#3777), EGFR (#4267), pRSK T359 (#8753), RSK1 (#9333), pMEK1/2 S217/S221 (#9154), pERK1/2 T202/Y204 (#4370), ERK1/2 (# 4696), pS6 S240/244 (“pS6”; #5364), rpS6 (“S6”; #2317), pAKT S473 (#4060), AKT (#2920), Rb (#9309), pMET Y1234/1235 (#3077), MET (#8198) (all from Cell Signaling Technology), BRAF (Santa Cruz Biotechnology, Inc. #sc-9002), CRAF (Bethyl Labs #A301-519A), pRb S780 (BD Biosciences #558385), and α-Tubulin (Sigma #T5168) were diluted in 5% BSA in 1x TBS-T.

    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: Antibodies against ARAF (#4432), pBRAF S445 (#2696), pCRAF S338 (#9427), pEGFR Y1068 (#3777), EGFR (#4267), pRSK T359 (#8753), RSK1 (#9333), pMEK1/2 S217/S221 (#9154), pERK1/2 T202/Y204 (#4370), ERK1/2 (# 4696), pS6 S240/244 (“pS6”; #5364), rpS6 (“S6”; #2317), pAKT S473 (#4060), AKT (#2920), Rb (#9309), pMET Y1234/1235 (#3077), MET (#8198) (all from Cell Signaling Technology), BRAF (Santa Cruz Biotechnology, Inc. #sc-9002), CRAF (Bethyl Labs #A301-519A), pRb S780 (BD Biosciences #558385), and α-Tubulin (Sigma #T5168) were diluted in 5% BSA in 1x TBS-T.

    Techniques: Staining, Flow Cytometry, Cytometry, Immunofluorescence

    Immunohistochemical staining results for pERK1/2 (×10). Staining is performed on whisker from the Abcc6 knockout mouse (A-C) and PXE skin tissues (D-F) . Alizarin Red stains was used to localize mineralization (A and D) . Positive staining for pERK1/2 is obtained on murine whisker ( B, arrow) and PXE skin ( E, asterisk), co-localizing with mineralization. The pERK1/2 staining being broader than the mineralization staining could represent the active mineralization process where pERK1/2 expression precedes calcification. No staining is noted in control whisker (C) and control skin tissue (F) . (n = 5 each). Scale bar = 100 μm.

    Journal: Orphanet Journal of Rare Diseases

    Article Title: Perturbation of specific pro-mineralizing signalling pathways in human and murine pseudoxanthoma elasticum

    doi: 10.1186/1750-1172-9-66

    Figure Lengend Snippet: Immunohistochemical staining results for pERK1/2 (×10). Staining is performed on whisker from the Abcc6 knockout mouse (A-C) and PXE skin tissues (D-F) . Alizarin Red stains was used to localize mineralization (A and D) . Positive staining for pERK1/2 is obtained on murine whisker ( B, arrow) and PXE skin ( E, asterisk), co-localizing with mineralization. The pERK1/2 staining being broader than the mineralization staining could represent the active mineralization process where pERK1/2 expression precedes calcification. No staining is noted in control whisker (C) and control skin tissue (F) . (n = 5 each). Scale bar = 100 μm.

    Article Snippet: Immunohistological analysis Immunohistochemistry (IHC) was performed on formalin-fixed and paraffin embedded whisker and eye tissues (5 μm) of Abcc6 KO mice and human dermal tissues prepared from lesional skin biopsies, using primary antibodies against BMP2 (Abcam, USA), pSMAD1 (Abcam, USA), pSMAD2 (Cell Signalling Technology, The Netherlands), pSMAD4, 5 (Abcam, USA) and pSMAD8 (Santa Cruz Biotechnology, Inc., Germany), pSMAD1-5-8 (an antibody recognizing SMAD1 only when dually phosphorylated at Ser463 and Ser465, as well as phosphorylated SMAD5 and SMAD8; Abcam, USA), RUNX2 (M70, Santa Cruz Biotechnology, Inc., Germany), CTGF (Abcam, UK), Caspase 3 (BIOKE, Cell Signalling Technology, The Netherlands) and pERK1/2 (Abcam, UK).

    Techniques: Immunohistochemistry, Staining, Whisker Assay, Knock-Out, Expressing

    p38, JNK, and ERK1/2 activation regulated by NaO in S. aureus -challenged bMECs . MAPK phosphorylation was measured in bMECs that were treated with 0.25 or 1 mM NaO and/or challenged with S. aureus by flow cytometry. The phosphorylated MAPK concentrations (U/ml) are represented: (A) pp38, (B) pJNK1/2, and (C) pERK1/2. Each bar shows the result of one experiment. SB203580: p38 inhibitor. SP600125: JNK1/2 inhibitor. U0126: ERK1/2 inhibitor. Different letters above the bars indicate significant changes among the unchallenged bMECs (white bars) and S. aureus -challenged cells within the same MAPK evaluated ( p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Sodium Octanoate Modulates the Innate Immune Response of Bovine Mammary Epithelial Cells through the TLR2/P38/JNK/ERK1/2 Pathway: Implications during Staphylococcus aureus Internalization

    doi: 10.3389/fcimb.2017.00078

    Figure Lengend Snippet: p38, JNK, and ERK1/2 activation regulated by NaO in S. aureus -challenged bMECs . MAPK phosphorylation was measured in bMECs that were treated with 0.25 or 1 mM NaO and/or challenged with S. aureus by flow cytometry. The phosphorylated MAPK concentrations (U/ml) are represented: (A) pp38, (B) pJNK1/2, and (C) pERK1/2. Each bar shows the result of one experiment. SB203580: p38 inhibitor. SP600125: JNK1/2 inhibitor. U0126: ERK1/2 inhibitor. Different letters above the bars indicate significant changes among the unchallenged bMECs (white bars) and S. aureus -challenged cells within the same MAPK evaluated ( p

    Article Snippet: MAPK activation during NaO-modulated S. aureus internalization by flow cytometry To evaluate the MAPK activation levels by flow cytometry, the bMECs were treated with 0.25 or 1 mM NaO (24 h), S. aureus or both, and the samples (30 μg of protein) were prepared according to the manufacturer's protocol for adherent cells (Becton Dickinson, Germany). pp38 (T180/Y182), pJNK1/2 (T183/185), and pERK1/2 (T202/Y204) were quantitatively determined using antibodies from a Flex Set Cytometric Bead Array (Becton Dickinson) according to the manufacturer's protocol.

    Techniques: Activation Assay, Flow Cytometry, Cytometry

    Involvement of ERK1/2 MAPK pathway in Tat-mediated upregulation of K V 1.3 expression. A : Western blot analysis for ERK1/2 MAPK. Microglia were exposed to 200 ng/ml of Tat and harvested at indicated times. Protein expression was analyzed by immunoblot using antibodies against ERK1/2 phosphorylation (pERK1/2) and total ERK1/2 (ERK1/2) MAPK. Tat up-regulated pERK1/2 MAPK in a time window from 30 min to 5 hr. B : K v channel antagonists inhibit ERK1/2 MAPK phosphorylation. Microglia were treated with MgTx (5 nM), PAP (10 nM), or 4-AP (1 mM) for 30 min followed by Tat at 200 ng/ml for additional 5 hr. Gel blots reveal a reduction of Tat-induced ERK1/2 phosphorylation in microglia treated with MgTx, PAP, or 4-AP, indicating a link between K v 1.3 channel activation and ERK1/2 MAPK signal pathway. C : Western blot results showing that the blockade of Tat enhancement of K v 1.3 expression in microglia was blocked by U0126, an inhibitor for MEK1 and MEK 2, further demonstrating the link between ERK1/2 MAPK and Tat-induced increase of K v 1.3 expression. D : TUNEL staining exhibited a significant increase of neuronal apoptosis induced by the supernatants collected from Tat-treated microglia and its blockade by U0126, a MEK1 and MEK2 inhibitor. Data were from three independent experiments. *** p

    Journal: PLoS ONE

    Article Title: HIV-1 Tat Protein Increases Microglial Outward K+ Current and Resultant Neurotoxic Activity

    doi: 10.1371/journal.pone.0064904

    Figure Lengend Snippet: Involvement of ERK1/2 MAPK pathway in Tat-mediated upregulation of K V 1.3 expression. A : Western blot analysis for ERK1/2 MAPK. Microglia were exposed to 200 ng/ml of Tat and harvested at indicated times. Protein expression was analyzed by immunoblot using antibodies against ERK1/2 phosphorylation (pERK1/2) and total ERK1/2 (ERK1/2) MAPK. Tat up-regulated pERK1/2 MAPK in a time window from 30 min to 5 hr. B : K v channel antagonists inhibit ERK1/2 MAPK phosphorylation. Microglia were treated with MgTx (5 nM), PAP (10 nM), or 4-AP (1 mM) for 30 min followed by Tat at 200 ng/ml for additional 5 hr. Gel blots reveal a reduction of Tat-induced ERK1/2 phosphorylation in microglia treated with MgTx, PAP, or 4-AP, indicating a link between K v 1.3 channel activation and ERK1/2 MAPK signal pathway. C : Western blot results showing that the blockade of Tat enhancement of K v 1.3 expression in microglia was blocked by U0126, an inhibitor for MEK1 and MEK 2, further demonstrating the link between ERK1/2 MAPK and Tat-induced increase of K v 1.3 expression. D : TUNEL staining exhibited a significant increase of neuronal apoptosis induced by the supernatants collected from Tat-treated microglia and its blockade by U0126, a MEK1 and MEK2 inhibitor. Data were from three independent experiments. *** p

    Article Snippet: PVDF membranes were then blocked with 5% dry milk in Tris-Buffered Saline (TBS) (all products from Bio-Rad Laboratories, Hercules, CA) and probed overnight at 4°C with either rabbit polyclonal KV 1.3 (1∶100; Alomone Lab, Israel), phospho-p44/42 MAPK (pERK1/2), total p44/42 MAPK (ERK1/2) (1∶1000; Cell Signaling Technology, Danvers, MA), or anti-mouse β-actin monoclonal antibody (1∶10,000, Sigma-Aldrich) primary Abs.

    Techniques: Expressing, Western Blot, Activation Assay, TUNEL Assay, Staining

    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

    ( a ) HCC827 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed by immuno-blotting using anti-pERK1/2, anti-pAkt, anti-pSrc and, anti-pEGFR (Tyr1068). Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, utilising Erk1/2, Akt, Src, EGFR and anti-tubulin antibodies. ( b ) H1975 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed as before. Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, as before

    Journal: Cell Communication and Signaling : CCS

    Article Title: USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells

    doi: 10.1186/s12964-018-0291-5

    Figure Lengend Snippet: ( a ) HCC827 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed by immuno-blotting using anti-pERK1/2, anti-pAkt, anti-pSrc and, anti-pEGFR (Tyr1068). Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, utilising Erk1/2, Akt, Src, EGFR and anti-tubulin antibodies. ( b ) H1975 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed as before. Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, as before

    Article Snippet: The following primary antibodies were used: rat anti-tubulin (1:10000, Abcam, Cambridge, UK), mouse anti-ERK, mouse anti-pERK1/2 (Thr202/Tyr204), rabbit anti-Src, rabbit anti-pSrc (Tyr416), rabbit anti-AKT, rabbit anti-pAKT (Ser473), rabbit anti-pEGFR (Tyr1068), rabbit anti-cleaved PARP, rabbit anti-cleaved caspase 3, mouse anti-caspase 9, rabbit anti-BCL-2 (1:1000, Cell Signaling, Danvers, USA), mouse anti-EGFR (BD Biosciences, USA).

    Techniques: Transfection

    DEP-1 regulates FL-stimulated FLT3 signaling. DEP-1 expression was stably downregulated in THP-1 cells by lentiviral transduction of shRNA. Control cells harbor a non-targeting shRNA construct. ( A ) FL-dependent ERK1/2 activation was assessed by immunoblotting of cell lysate samples with antibodies recognizing activated ERK1/2 (pERK1/2). Loading was analyzed by reblot with ERK1/2 antibodies. Representative experiment; the efficiency of stable DEP-1 knockdown is also shown (upper panel). ( B ) Quantitative data for 4 independent experiments. Numbers represent pERK1/2 signals normalized to ERK1/2 levels in the same sample. The value of control cells at 10 min was set to 1.0. Significance for the difference between responses of the two different cell pools was determined by two-way ANOVA.

    Journal: PLoS ONE

    Article Title: Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay

    doi: 10.1371/journal.pone.0062871

    Figure Lengend Snippet: DEP-1 regulates FL-stimulated FLT3 signaling. DEP-1 expression was stably downregulated in THP-1 cells by lentiviral transduction of shRNA. Control cells harbor a non-targeting shRNA construct. ( A ) FL-dependent ERK1/2 activation was assessed by immunoblotting of cell lysate samples with antibodies recognizing activated ERK1/2 (pERK1/2). Loading was analyzed by reblot with ERK1/2 antibodies. Representative experiment; the efficiency of stable DEP-1 knockdown is also shown (upper panel). ( B ) Quantitative data for 4 independent experiments. Numbers represent pERK1/2 signals normalized to ERK1/2 levels in the same sample. The value of control cells at 10 min was set to 1.0. Significance for the difference between responses of the two different cell pools was determined by two-way ANOVA.

    Article Snippet: The following antibodies were used: Rabbit polyclonal anti-FLT3 antibodies S18 (SC-480) from Santa Cruz Biotechnology (Heidelberg, Germany), polyclonal goat anti-DEP-1 (AF1934) antibodies from R & D Systems (Wiesbaden, Germany), rabbit monoclonal anti-pERK1/2 (#4695) and rabbit monoclonal anti-ERK1/2 (#4695) from Cell Signaling (Frankfurt, Germany) and FITC-labeled anti-rabbit IgG from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

    Techniques: Expressing, Stable Transfection, Transduction, shRNA, Construct, Activation Assay

    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