phospho erk 1 2  (Cell Signaling Technology Inc)

 
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  • 98
    Name:
    PTEN Blocking Peptide
    Description:
    This peptide is used to block PTEN D4 3 XP Rabbit mAb 9188 reactivity
    Catalog Number:
    1250
    Price:
    None
    Category:
    Experimental Controls
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    Structured Review

    Cell Signaling Technology Inc phospho erk 1 2
    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
    This peptide is used to block PTEN D4 3 XP Rabbit mAb 9188 reactivity
    https://www.bioz.com/result/phospho erk 1 2/product/Cell Signaling Technology Inc
    Average 98 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    phospho erk 1 2 - by Bioz Stars, 2020-11
    98/100 stars

    Images

    1) Product Images from "ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury"

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    Journal: Neurochemical Research

    doi: 10.1007/s11064-018-2558-z

    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition. a and b Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Techniques Used: Activation Assay, Activity Assay, Inhibition, Western Blot, Cell Culture

    The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery
    Figure Legend Snippet: The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Techniques Used: Activity Assay, Functional Assay

    BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals.  a  Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126.  b  Quantification analysis of the infarct volume [n = 6, *P 
    Figure Legend Snippet: BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals. a Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126. b Quantification analysis of the infarct volume [n = 6, *P 

    Techniques Used: Inhibition, Activation Assay, Staining

    BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation.  a  Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 
    Figure Legend Snippet: BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation. a Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Techniques Used: Functional Assay, Inhibition, Activation Assay, Injection

    After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased.  a  and  b  Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm.  c  and  d  Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 
    Figure Legend Snippet: After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased. a and b Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm. c and d Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Techniques Used: Double Immunofluorescence Staining, Western Blot, Expressing

    BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury.  a  A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure.  b  and  c  Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 
    Figure Legend Snippet: BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury. a A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure. b and c Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Techniques Used: Western Blot, Expressing

    BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level.  a  Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level. a Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Techniques Used: Activity Assay, Western Blot, Expressing

    2) Product Images from "Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning"

    Article Title: Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00393

    Heparan sulfate induces an apoptosis/necroptosis signal pathway.  (A)  HS fragments are cleaved by heparanase from a HS proteoglycan, which is localized on the plasma membrane of endothelial cells.  (B)  Structure of HS proteoglycan.  (C)  HS interacts with a pattern recognition receptor (i.e., toll-like receptor 4) localized on the cell surface of cardiomyocytes and activates a pro-apoptotic intrinsic pathway. The signaling cascade involves phosphorylation of ERK 1/2 resulting in the release of mitochondrial cytochrome C, which leads to cleavage and activation of caspase 3. In the next step of this pathway, PARP is cleaved and deactivated by activated caspase 3. Induction of TNF-α caused by HS inhibits the pro-apoptotic pathway and induces phosphorylation of RIP3 and necroptosis. These apoptosis and necroptosis signal pathways represent our Petri net model. White circles are places, and black rectangles are transitions. Unidirectional arcs indicate directed flows. Bidirectional arcs indicate read arcs, which influence transitions, but do not consume tokens. Modified from Martin et al., Sarrazin et al., and Maeda (  13 –  15 ). HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; ERK, extracellular signal-regulated kinase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha; Ser, serine; Xyl, xylose; Gal, galactose; GlcNAc, N-acetylgalactosamine; IdoA, iduronic acid.
    Figure Legend Snippet: Heparan sulfate induces an apoptosis/necroptosis signal pathway. (A) HS fragments are cleaved by heparanase from a HS proteoglycan, which is localized on the plasma membrane of endothelial cells. (B) Structure of HS proteoglycan. (C) HS interacts with a pattern recognition receptor (i.e., toll-like receptor 4) localized on the cell surface of cardiomyocytes and activates a pro-apoptotic intrinsic pathway. The signaling cascade involves phosphorylation of ERK 1/2 resulting in the release of mitochondrial cytochrome C, which leads to cleavage and activation of caspase 3. In the next step of this pathway, PARP is cleaved and deactivated by activated caspase 3. Induction of TNF-α caused by HS inhibits the pro-apoptotic pathway and induces phosphorylation of RIP3 and necroptosis. These apoptosis and necroptosis signal pathways represent our Petri net model. White circles are places, and black rectangles are transitions. Unidirectional arcs indicate directed flows. Bidirectional arcs indicate read arcs, which influence transitions, but do not consume tokens. Modified from Martin et al., Sarrazin et al., and Maeda ( 13 – 15 ). HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; ERK, extracellular signal-regulated kinase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha; Ser, serine; Xyl, xylose; Gal, galactose; GlcNAc, N-acetylgalactosamine; IdoA, iduronic acid.

    Techniques Used: Activation Assay, Modification

    Simulated time course of all involved components of the apoptosis/necroptosis signal pathway induced by HS. Simulated time course of the components involved in intrinsic apoptosis and necroptosis signaling pathways for cardiomyocytes exposed to three different HS concentrations [5 (green), 10 (blue), and 20 (purple) μg/ml of HS]. The blue background represents the standard error of the measured data. The model was simulated based on relative cleaved PARP values. The filled points in graphs represent data used for training and the unfilled for validation. Simulated time course for relative protein expression of  (A)  phosphorylation of ERK 1/2,  (B)  cytochrome C,  (C)  cleaved caspase 3,  (D)  cleaved PARP, and  (E)  phosphorylation of RIP3 is shown.  (F)  Simulated data of relative TNF-α mRNA expression. HS, heparan sulfate; ph-ERK, phospho-extracellular signal-regulated kinase; PARP, poly-(ADP-ribose) polymerase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha.
    Figure Legend Snippet: Simulated time course of all involved components of the apoptosis/necroptosis signal pathway induced by HS. Simulated time course of the components involved in intrinsic apoptosis and necroptosis signaling pathways for cardiomyocytes exposed to three different HS concentrations [5 (green), 10 (blue), and 20 (purple) μg/ml of HS]. The blue background represents the standard error of the measured data. The model was simulated based on relative cleaved PARP values. The filled points in graphs represent data used for training and the unfilled for validation. Simulated time course for relative protein expression of (A) phosphorylation of ERK 1/2, (B) cytochrome C, (C) cleaved caspase 3, (D) cleaved PARP, and (E) phosphorylation of RIP3 is shown. (F) Simulated data of relative TNF-α mRNA expression. HS, heparan sulfate; ph-ERK, phospho-extracellular signal-regulated kinase; PARP, poly-(ADP-ribose) polymerase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha.

    Techniques Used: Expressing

    Heparan sulfate induces a pro-apoptotic pathway. HL-1 cells exposed to 10 μg/ml HS for 16 h showed a significant increase in protein expression of  (A)  phospho-ERK 1/2,  (B)  cytochrome C,  (C) cleaved  PARP, and  (D) cleaved  caspase 3, compared to unstimulated cells. Protein expression was normalized to unstimulated cells.  (E)  Relative mRNA expressions of HL-1 cells exposed to HS were analyzed by quantitative real-time PCR, compared to unstimulated cells. Caspase 3 mRNA expression was normalized to reference gene S7 and unstimulated cells.  (F)  Relative caspase 3 activity of cardiomyocytes exposed to HS, compared to unstimulated cells. The data represent the mean ± SD of triplicate samples for three independent experiments. HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; p-ERK, phospho-extracellular signal-regulated kinase; statistical significance was performed by using unpaired  t -test. * p
    Figure Legend Snippet: Heparan sulfate induces a pro-apoptotic pathway. HL-1 cells exposed to 10 μg/ml HS for 16 h showed a significant increase in protein expression of (A) phospho-ERK 1/2, (B) cytochrome C, (C) cleaved PARP, and (D) cleaved caspase 3, compared to unstimulated cells. Protein expression was normalized to unstimulated cells. (E) Relative mRNA expressions of HL-1 cells exposed to HS were analyzed by quantitative real-time PCR, compared to unstimulated cells. Caspase 3 mRNA expression was normalized to reference gene S7 and unstimulated cells. (F) Relative caspase 3 activity of cardiomyocytes exposed to HS, compared to unstimulated cells. The data represent the mean ± SD of triplicate samples for three independent experiments. HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; p-ERK, phospho-extracellular signal-regulated kinase; statistical significance was performed by using unpaired t -test. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activity Assay

    3) Product Images from "ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell"

    Article Title: ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.10564

    (A) LDIR induced radio-resistance in A549 detected by clonogenic assay. Cells were either pre-irradiated with 5cGy α particle or 0Gy α particle. 6 hours later, these cells were irradiated with 75cGy α particle. 6 hours after the 75cGy radiation, clonogenic assay was used to determine the survival fraction. Sham represented non-irradiated group. (B) Detection of apoptosis related proteins and pH 2 AX(Ser139) by WB. Radiation treatment steps were same as above mentioned. 3 hours after the 75cGy radiation, cell samples were collected for WB analysis of pH 2 AX(Ser139). 24 hours after the 75cGy radiation, cell samples were collected for WB analysis of Bax and Bcl-2. (C) Activation of ERK 1/2 in LDIR induced radio-resistance. Detection of phosphor-ERK 1/2 by WB was performed 24 hours after the 75cGy radiation. To evaluate the effect of ERK 1/2 inhibitor, final concentration of 5μM U0126 was added into the culture medium 30 minutes before 5cGy radiation. (D) Confirmation of ERK 1/2 activation in LDIR induced radio-resistance by clonogenic assay. Final concentration of 5μM U0126 was added into the culture medium 30 minutes before 5cGy radiation. 6 hours after 5cGy radiation, cells were irradiated with 75cGy. And after 6 hours incubation, clonogenic assay was performed to determine the survival fraction.
    Figure Legend Snippet: (A) LDIR induced radio-resistance in A549 detected by clonogenic assay. Cells were either pre-irradiated with 5cGy α particle or 0Gy α particle. 6 hours later, these cells were irradiated with 75cGy α particle. 6 hours after the 75cGy radiation, clonogenic assay was used to determine the survival fraction. Sham represented non-irradiated group. (B) Detection of apoptosis related proteins and pH 2 AX(Ser139) by WB. Radiation treatment steps were same as above mentioned. 3 hours after the 75cGy radiation, cell samples were collected for WB analysis of pH 2 AX(Ser139). 24 hours after the 75cGy radiation, cell samples were collected for WB analysis of Bax and Bcl-2. (C) Activation of ERK 1/2 in LDIR induced radio-resistance. Detection of phosphor-ERK 1/2 by WB was performed 24 hours after the 75cGy radiation. To evaluate the effect of ERK 1/2 inhibitor, final concentration of 5μM U0126 was added into the culture medium 30 minutes before 5cGy radiation. (D) Confirmation of ERK 1/2 activation in LDIR induced radio-resistance by clonogenic assay. Final concentration of 5μM U0126 was added into the culture medium 30 minutes before 5cGy radiation. 6 hours after 5cGy radiation, cells were irradiated with 75cGy. And after 6 hours incubation, clonogenic assay was performed to determine the survival fraction.

    Techniques Used: Clonogenic Assay, Irradiation, Western Blot, Activation Assay, Concentration Assay, Incubation

    4) Product Images from "Genetic targeting of B-RafV600E affects survival and proliferation and identifies selective agents against BRAF-mutant colorectal cancer cells"

    Article Title: Genetic targeting of B-RafV600E affects survival and proliferation and identifies selective agents against BRAF-mutant colorectal cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-122

    Chemosensitivity for B-Raf inhibitors. A : Multi-kinase inhibitor sorafenib had no differential effect on RKO  BRAF  knockout cell clones.  B-C : Although no significant differences in the IC 50  were observed, by trend RBW-1 was less sensitive to vemurafenib and RAF265 compared to cells with mutant  BRAF  alleles.  D : The IC 50  of the B-Raf kinase inhibitor dabrafenib in RBW-1 was 5.3 times higher than the IC 50  of the parental clones RKO-E1 and RBOW, and 20 times higher than in clones carrying a  BRAF -mutant allele only.  E : Aliquots of each cell line were incubated with the respective IC 50  concentrations of B-Raf inhibitors, lyzed, and analyzed in Western blots for phosphorylation of Mek 1/2 (upper panel) and Erk 1/2 (lower panel).  F : Signal intensities of phospho-Mek 1/2 and phosphor-Erk 1/2 were normalized to the total fractions in densitometric analyses.
    Figure Legend Snippet: Chemosensitivity for B-Raf inhibitors. A : Multi-kinase inhibitor sorafenib had no differential effect on RKO BRAF knockout cell clones. B-C : Although no significant differences in the IC 50 were observed, by trend RBW-1 was less sensitive to vemurafenib and RAF265 compared to cells with mutant BRAF alleles. D : The IC 50 of the B-Raf kinase inhibitor dabrafenib in RBW-1 was 5.3 times higher than the IC 50 of the parental clones RKO-E1 and RBOW, and 20 times higher than in clones carrying a BRAF -mutant allele only. E : Aliquots of each cell line were incubated with the respective IC 50 concentrations of B-Raf inhibitors, lyzed, and analyzed in Western blots for phosphorylation of Mek 1/2 (upper panel) and Erk 1/2 (lower panel). F : Signal intensities of phospho-Mek 1/2 and phosphor-Erk 1/2 were normalized to the total fractions in densitometric analyses.

    Techniques Used: Knock-Out, Clone Assay, Mutagenesis, Incubation, Western Blot

    Verification of results in independent  BRAF  knockout cells. A : Cell death was assessed by determining the sub G1-fraction by flow cytometry and analyzed on a molecular level by Western blotting for PUMA.  B-D : In chemosensitivity assays RAF265 and vemurafenib did not induce different responses, while with dabrafenib, the wild type clone showed a 2.6 fold higher IC 50  value as compared to control clones.  E-F : In Western blotting experiments and subsequent densitometry analysis, IC 50  concentrations of dabrafenib showed a stronger effect on relative phosphorylation levels of Mek 1/2 and Erk 1/2 in parental and  BRAF -mutant cells than RAF265 and vemurafenib.  G : In proliferation assays, no differential responses were observed for PI-103.  H :  PIK3CA  status was confirmed by sequencing.  I : Phosphorylation of AKT was examined by Western blotting.
    Figure Legend Snippet: Verification of results in independent BRAF knockout cells. A : Cell death was assessed by determining the sub G1-fraction by flow cytometry and analyzed on a molecular level by Western blotting for PUMA. B-D : In chemosensitivity assays RAF265 and vemurafenib did not induce different responses, while with dabrafenib, the wild type clone showed a 2.6 fold higher IC 50 value as compared to control clones. E-F : In Western blotting experiments and subsequent densitometry analysis, IC 50 concentrations of dabrafenib showed a stronger effect on relative phosphorylation levels of Mek 1/2 and Erk 1/2 in parental and BRAF -mutant cells than RAF265 and vemurafenib. G : In proliferation assays, no differential responses were observed for PI-103. H : PIK3CA status was confirmed by sequencing. I : Phosphorylation of AKT was examined by Western blotting.

    Techniques Used: Knock-Out, Flow Cytometry, Cytometry, Western Blot, Clone Assay, Mutagenesis, Sequencing

    5) Product Images from "Mouse Cytomegalovirus M33 Is Necessary and Sufficient in Virus-Induced Vascular Smooth Muscle Cell Migration"

    Article Title: Mouse Cytomegalovirus M33 Is Necessary and Sufficient in Virus-Induced Vascular Smooth Muscle Cell Migration

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.16.10788-10795.2005

    RANTES stimulation of M33 activates Rac1. AoSMCs expressing M33 and/or trans were stimulated with 10 ng/ml recombinant mRANTES. Lysates were baited with GST-CRIB for active Rac1 pull-down assays. Active and total Rac1 were determined by immunoblotting using a Rac1-specific antibody. ERK-1/2 phosphorylation/activation was determined by Western blotting using phosphospecific antibodies. Total input protein was detected by Western blotting for Rac1 and ERK-1/2. The relative levels of active Rac1 and ERK-1/2 were normalized to the total input protein using NIH ImageJ. p-ERK, phospho-ERK.
    Figure Legend Snippet: RANTES stimulation of M33 activates Rac1. AoSMCs expressing M33 and/or trans were stimulated with 10 ng/ml recombinant mRANTES. Lysates were baited with GST-CRIB for active Rac1 pull-down assays. Active and total Rac1 were determined by immunoblotting using a Rac1-specific antibody. ERK-1/2 phosphorylation/activation was determined by Western blotting using phosphospecific antibodies. Total input protein was detected by Western blotting for Rac1 and ERK-1/2. The relative levels of active Rac1 and ERK-1/2 were normalized to the total input protein using NIH ImageJ. p-ERK, phospho-ERK.

    Techniques Used: Expressing, Recombinant, Activation Assay, Western Blot

    6) Product Images from "Lysosomal Sequestration of Sunitinib: A Novel Mechanism of Drug Resistance"

    Article Title: Lysosomal Sequestration of Sunitinib: A Novel Mechanism of Drug Resistance

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-11-1667

    High intratumoral concentrations of sunitinib inhibit tumor cells directly. A, intratumoral and plasma concentrations of sunitinib in mice with RCC tumors ( n  = 3) and patients with advanced malignancies ( n  = 3), treated with sunitinib at a dose of 40 mg/kg/d for 1 month (mice) or 37.5 to 50 mg/d for at least 4 weeks (patients). B, proliferation (MTT assay) of tumor cell lines incubated with increasing sunitinib concentrations. Sunitinib inhibited proliferation of tumor cells at clinically relevant intratumoral concentrations. C, sunitinib inhibition of colony formation of tumor cell lines. D, proliferation of endothelial cells incubated with increasing concentrations of sunitinib while grown in complete medium. HUVECs were cultured in M199 medium supplemented with 10% FBS and 10% human serum, whereas ECFCs were cultured in endothelial basal medium (EBM-2) supplemented with 10% FBS, VEGF, epidermal growth factor (EGF), insulin—like growth factor (IGF), and FGF. E, Western blot analysis of (phosphorylated) ERK 1/2 and Akt in 786-O and HT-29 cell lines treated with 2 μmol/L sunitinib for 1 hour, compared with DMSO controls. Results are shown as means ± SEM. *,  P
    Figure Legend Snippet: High intratumoral concentrations of sunitinib inhibit tumor cells directly. A, intratumoral and plasma concentrations of sunitinib in mice with RCC tumors ( n = 3) and patients with advanced malignancies ( n = 3), treated with sunitinib at a dose of 40 mg/kg/d for 1 month (mice) or 37.5 to 50 mg/d for at least 4 weeks (patients). B, proliferation (MTT assay) of tumor cell lines incubated with increasing sunitinib concentrations. Sunitinib inhibited proliferation of tumor cells at clinically relevant intratumoral concentrations. C, sunitinib inhibition of colony formation of tumor cell lines. D, proliferation of endothelial cells incubated with increasing concentrations of sunitinib while grown in complete medium. HUVECs were cultured in M199 medium supplemented with 10% FBS and 10% human serum, whereas ECFCs were cultured in endothelial basal medium (EBM-2) supplemented with 10% FBS, VEGF, epidermal growth factor (EGF), insulin—like growth factor (IGF), and FGF. E, Western blot analysis of (phosphorylated) ERK 1/2 and Akt in 786-O and HT-29 cell lines treated with 2 μmol/L sunitinib for 1 hour, compared with DMSO controls. Results are shown as means ± SEM. *, P

    Techniques Used: Mouse Assay, MTT Assay, Incubation, Inhibition, Cell Culture, Western Blot

    7) Product Images from "Mechanical Stretch Increases the Proliferation While Inhibiting the Osteogenic Differentiation in Dental Pulp Stem Cells"

    Article Title: Mechanical Stretch Increases the Proliferation While Inhibiting the Osteogenic Differentiation in Dental Pulp Stem Cells

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2012.0099

    Phosphorylation in response to uniaxial stretch for the indicated periods in DPSCs.  (A)  Stretch-stimulated phosphorylation of Akt ( n =4).  (B)  Stretch-stimulated phosphorylation of p38 MAPK ( n =4).  (C)  Stretch-stimulated phosphorylation of ERK 1/2. Results
    Figure Legend Snippet: Phosphorylation in response to uniaxial stretch for the indicated periods in DPSCs. (A) Stretch-stimulated phosphorylation of Akt ( n =4). (B) Stretch-stimulated phosphorylation of p38 MAPK ( n =4). (C) Stretch-stimulated phosphorylation of ERK 1/2. Results

    Techniques Used:

    8) Product Images from "Repeated Swim-Stress Induces Kappa Opioid-Mediated Activation of ERK1/2 MAPK"

    Article Title: Repeated Swim-Stress Induces Kappa Opioid-Mediated Activation of ERK1/2 MAPK

    Journal: Neuroreport

    doi: 10.1097/WNR.0b013e32830dd655

    Repeated swim-stress exposure induces KOR-dependent phospho-ERK1/2-ir in the mouse striatum. (a) Representative western blot of P-ERK1/2 (p44/p42) in wild-type mouse striatum after a single (Acute Swim) forced swim stress (S) in the presence or absence of the kappa-opioid antagonist norBNI (10 mg/kg, given 1hr prior to FSS) (b) Representative western blot of P-ERK1/2 in wild-type mouse striatum after repeated S (Repeated Swim), in the presence (S+norBNI) or absence (S) of norBNI. (c) Representative western blot of P-ERK1/2 time course following repeated S. Blots show P-ERK1/2 levels 10 min, 30 min, and 60 minutes after stress. (d) Representative western blot of P-ERK1/2 time course following repeated S in animals pretreated with the KOR-antagonist, norBNI (10mg/kg, 1hr). Below each blot of the P-ERK1/2 image is the protein loading control showing similar levels of β-actin. (e) Data are the mean ± S.E.M. of the quantified band intensities taken from repeated swim stress-induced P-ERK 1/2 activity in WT mice (KOR +/+), mice pretreated with norBNI (10mg/kg), or mice lacking KOR (KOR −/−) compared with the saline-treated no swim-stress comparable group (basal; dashed line). * p
    Figure Legend Snippet: Repeated swim-stress exposure induces KOR-dependent phospho-ERK1/2-ir in the mouse striatum. (a) Representative western blot of P-ERK1/2 (p44/p42) in wild-type mouse striatum after a single (Acute Swim) forced swim stress (S) in the presence or absence of the kappa-opioid antagonist norBNI (10 mg/kg, given 1hr prior to FSS) (b) Representative western blot of P-ERK1/2 in wild-type mouse striatum after repeated S (Repeated Swim), in the presence (S+norBNI) or absence (S) of norBNI. (c) Representative western blot of P-ERK1/2 time course following repeated S. Blots show P-ERK1/2 levels 10 min, 30 min, and 60 minutes after stress. (d) Representative western blot of P-ERK1/2 time course following repeated S in animals pretreated with the KOR-antagonist, norBNI (10mg/kg, 1hr). Below each blot of the P-ERK1/2 image is the protein loading control showing similar levels of β-actin. (e) Data are the mean ± S.E.M. of the quantified band intensities taken from repeated swim stress-induced P-ERK 1/2 activity in WT mice (KOR +/+), mice pretreated with norBNI (10mg/kg), or mice lacking KOR (KOR −/−) compared with the saline-treated no swim-stress comparable group (basal; dashed line). * p

    Techniques Used: Western Blot, Activity Assay, Mouse Assay

    Repeated swim-stress results in KOR-dependent phospho-ERK 1/2 (P-ERK 1/2) staining in mouse caudate (CPu) and nucleus accumbens (NAc). (a) Representative images of P-ERK 1/2 in CPu min after repeated swim-stress exposure (60 min), in stressed or nonstressed KOR wild-type (KOR +/+) and stressed KOR knockout mice (KOR −/−). (b) Representative images of P-ERK1/2-ir in NAc after repeated swim-stress exposure (60 min), in stressed or nonstressed KOR (+/+), stressed KOR (−/−) mice. (c) Higher power representative image of P-ERK1/2 staining in mouse NAc following repeated swim-stress exposure and stressed mice that were pretreated with the selective kappa opioid antagonist, norbinaltorphimine (norBNI, 10mg/kg i.p., 1hr prior to stress) All images representative of 3 independent experiments.
    Figure Legend Snippet: Repeated swim-stress results in KOR-dependent phospho-ERK 1/2 (P-ERK 1/2) staining in mouse caudate (CPu) and nucleus accumbens (NAc). (a) Representative images of P-ERK 1/2 in CPu min after repeated swim-stress exposure (60 min), in stressed or nonstressed KOR wild-type (KOR +/+) and stressed KOR knockout mice (KOR −/−). (b) Representative images of P-ERK1/2-ir in NAc after repeated swim-stress exposure (60 min), in stressed or nonstressed KOR (+/+), stressed KOR (−/−) mice. (c) Higher power representative image of P-ERK1/2 staining in mouse NAc following repeated swim-stress exposure and stressed mice that were pretreated with the selective kappa opioid antagonist, norbinaltorphimine (norBNI, 10mg/kg i.p., 1hr prior to stress) All images representative of 3 independent experiments.

    Techniques Used: Staining, Knock-Out, Mouse Assay

    9) Product Images from "Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning"

    Article Title: Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00393

    Heparan sulfate induces a pro-apoptotic pathway. HL-1 cells exposed to 10 μg/ml HS for 16 h showed a significant increase in protein expression of  (A)  phospho-ERK 1/2,  (B)  cytochrome C,  (C) cleaved  PARP, and  (D) cleaved  caspase 3, compared to unstimulated cells. Protein expression was normalized to unstimulated cells.  (E)  Relative mRNA expressions of HL-1 cells exposed to HS were analyzed by quantitative real-time PCR, compared to unstimulated cells. Caspase 3 mRNA expression was normalized to reference gene S7 and unstimulated cells.  (F)  Relative caspase 3 activity of cardiomyocytes exposed to HS, compared to unstimulated cells. The data represent the mean ± SD of triplicate samples for three independent experiments. HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; p-ERK, phospho-extracellular signal-regulated kinase; statistical significance was performed by using unpaired  t -test. * p
    Figure Legend Snippet: Heparan sulfate induces a pro-apoptotic pathway. HL-1 cells exposed to 10 μg/ml HS for 16 h showed a significant increase in protein expression of (A) phospho-ERK 1/2, (B) cytochrome C, (C) cleaved PARP, and (D) cleaved caspase 3, compared to unstimulated cells. Protein expression was normalized to unstimulated cells. (E) Relative mRNA expressions of HL-1 cells exposed to HS were analyzed by quantitative real-time PCR, compared to unstimulated cells. Caspase 3 mRNA expression was normalized to reference gene S7 and unstimulated cells. (F) Relative caspase 3 activity of cardiomyocytes exposed to HS, compared to unstimulated cells. The data represent the mean ± SD of triplicate samples for three independent experiments. HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; p-ERK, phospho-extracellular signal-regulated kinase; statistical significance was performed by using unpaired t -test. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activity Assay

    Simulated time course of all involved components of the apoptosis/necroptosis signal pathway induced by HS. Simulated time course of the components involved in intrinsic apoptosis and necroptosis signaling pathways for cardiomyocytes exposed to three different HS concentrations [5 (green), 10 (blue), and 20 (purple) μg/ml of HS]. The blue background represents the standard error of the measured data. The model was simulated based on relative cleaved PARP values. The filled points in graphs represent data used for training and the unfilled for validation. Simulated time course for relative protein expression of  (A)  phosphorylation of ERK 1/2,  (B)  cytochrome C,  (C)  cleaved caspase 3,  (D)  cleaved PARP, and  (E)  phosphorylation of RIP3 is shown.  (F)  Simulated data of relative TNF-α mRNA expression. HS, heparan sulfate; ph-ERK, phospho-extracellular signal-regulated kinase; PARP, poly-(ADP-ribose) polymerase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha.
    Figure Legend Snippet: Simulated time course of all involved components of the apoptosis/necroptosis signal pathway induced by HS. Simulated time course of the components involved in intrinsic apoptosis and necroptosis signaling pathways for cardiomyocytes exposed to three different HS concentrations [5 (green), 10 (blue), and 20 (purple) μg/ml of HS]. The blue background represents the standard error of the measured data. The model was simulated based on relative cleaved PARP values. The filled points in graphs represent data used for training and the unfilled for validation. Simulated time course for relative protein expression of (A) phosphorylation of ERK 1/2, (B) cytochrome C, (C) cleaved caspase 3, (D) cleaved PARP, and (E) phosphorylation of RIP3 is shown. (F) Simulated data of relative TNF-α mRNA expression. HS, heparan sulfate; ph-ERK, phospho-extracellular signal-regulated kinase; PARP, poly-(ADP-ribose) polymerase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha.

    Techniques Used: Expressing

    10) Product Images from "Loss of Caveolin-1 Gene Expression Accelerates the Development of Dysplastic Mammary Lesions in Tumor-Prone Transgenic Mice"

    Article Title: Loss of Caveolin-1 Gene Expression Accelerates the Development of Dysplastic Mammary Lesions in Tumor-Prone Transgenic Mice

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-08-0503

    Loss of caveolin-1 dramatically up-regulates cyclin D1 expression in dysplastic mammary lesions. (A) Immunoblot analysis . Mammary glands were harvested from 4-wk-old PyMT/Cav-1 (−/−) and PyMT/Cav-1 (+/+) mice and homogenized in lysis buffer. Tissue lysates were prepared, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Normalization of epithelial cell content was performed using a pan-cytokeratin antibody. Relative levels of phospho-STAT5a were determined by immunoblotting with a phospho-specific antibody probe that selectively recognizes activated STAT5a at its Jak-2 phosphorylation site (pY694). Similarly, the levels of phospho-ERK-1/2 were determined by immunoblotting with a phospho-specific antibody probe that selectively recognizes activated ERK-1/2 (pT202/pY204). Phospho-independent anti-ERK-1/2 IgG and anti-STAT5a IgG were used as controls for equal loading. Cyclin D1 expression levels were monitored using a specific rabbit polyclonal antibody. Note that cyclin D1 protein expression is dramatically elevated in PyMT/Cav-1 (−/−) samples, compared with matched-samples derived from PyMT/Cav-1 (+/+) mice. However, the levels of total STAT5a and phospho-STAT5a remain unchanged in PyMT/Cav-1 (−/−) samples. Similarly, the levels of total ERK-1/2 and phospho-ERK-1/2 were not elevated in PyMT/Cav-1 (−/−) samples. (B) Immunohistochemistry. To visualize the cellular distribution of cyclin D1, we performed immunohistochemical analysis on dysplastic mammary lesions derived from 4-wk-old PyMT/Cav-1 (−/−) and PyMT/Cav-1 (+/+) mice. Paraffin-embedded mammary glands were sectioned at 5 μm and immunostained with a rabbit polyclonal antibody to cyclin D1. Lesions of approximately the same size from each genotype were chosen to allow for a better comparison. Representative examples are shown. Note that the intensity of cyclin D1 immunostaining (brown color) is clearly increased in the PyMT/Cav-1 (−/−) dysplastic mammary lesions and the nuclei of these dysplastic mammary epithelial cells are more densely stained. The cyclin D1 staining pattern was also noticeably altered in PyMT/Cav-1 (−/−) samples. In PyMT/Cav-1 (+/+) mammary lesions, cyclin D1 immunostaining was confined to the outermost layers of mammary epithelial cells; little or no staining was observed in the center of the lesion (see arrow). In contrast, in PyMT/Cav-1 (−/−) mammary lesions, cyclin D1 immunostaining was present in virtually all the epithelial cells and extended to the center of the lesion. No changes in cyclin D1 immunostaining were observed in the surrounding stromal cells.
    Figure Legend Snippet: Loss of caveolin-1 dramatically up-regulates cyclin D1 expression in dysplastic mammary lesions. (A) Immunoblot analysis . Mammary glands were harvested from 4-wk-old PyMT/Cav-1 (−/−) and PyMT/Cav-1 (+/+) mice and homogenized in lysis buffer. Tissue lysates were prepared, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Normalization of epithelial cell content was performed using a pan-cytokeratin antibody. Relative levels of phospho-STAT5a were determined by immunoblotting with a phospho-specific antibody probe that selectively recognizes activated STAT5a at its Jak-2 phosphorylation site (pY694). Similarly, the levels of phospho-ERK-1/2 were determined by immunoblotting with a phospho-specific antibody probe that selectively recognizes activated ERK-1/2 (pT202/pY204). Phospho-independent anti-ERK-1/2 IgG and anti-STAT5a IgG were used as controls for equal loading. Cyclin D1 expression levels were monitored using a specific rabbit polyclonal antibody. Note that cyclin D1 protein expression is dramatically elevated in PyMT/Cav-1 (−/−) samples, compared with matched-samples derived from PyMT/Cav-1 (+/+) mice. However, the levels of total STAT5a and phospho-STAT5a remain unchanged in PyMT/Cav-1 (−/−) samples. Similarly, the levels of total ERK-1/2 and phospho-ERK-1/2 were not elevated in PyMT/Cav-1 (−/−) samples. (B) Immunohistochemistry. To visualize the cellular distribution of cyclin D1, we performed immunohistochemical analysis on dysplastic mammary lesions derived from 4-wk-old PyMT/Cav-1 (−/−) and PyMT/Cav-1 (+/+) mice. Paraffin-embedded mammary glands were sectioned at 5 μm and immunostained with a rabbit polyclonal antibody to cyclin D1. Lesions of approximately the same size from each genotype were chosen to allow for a better comparison. Representative examples are shown. Note that the intensity of cyclin D1 immunostaining (brown color) is clearly increased in the PyMT/Cav-1 (−/−) dysplastic mammary lesions and the nuclei of these dysplastic mammary epithelial cells are more densely stained. The cyclin D1 staining pattern was also noticeably altered in PyMT/Cav-1 (−/−) samples. In PyMT/Cav-1 (+/+) mammary lesions, cyclin D1 immunostaining was confined to the outermost layers of mammary epithelial cells; little or no staining was observed in the center of the lesion (see arrow). In contrast, in PyMT/Cav-1 (−/−) mammary lesions, cyclin D1 immunostaining was present in virtually all the epithelial cells and extended to the center of the lesion. No changes in cyclin D1 immunostaining were observed in the surrounding stromal cells.

    Techniques Used: Expressing, Mouse Assay, Lysis, SDS Page, Derivative Assay, Immunohistochemistry, Immunostaining, Staining

    11) Product Images from "Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni"

    Article Title: Serine phosphorylation of cortactin is required for maximal host cell invasion by Campylobacter jejuni

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-11-82

    Model of  C. jejuni  internalization. C. jejuni  invasion of host cells. Step 1:  C. jejuni  binds to fibronectin (Fn) via the two  C. jejuni  Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [  43 ,  44 ] causing activation of the α 5 β 1  integrin receptors and the epidermal growth factor receptor (EGFR) [  27 ,  45 ]. Step 2: Activation of the α 5 β 1  integrin leads to the recruitment and partial activation of FAK and paxillin [  27 ,  46 ]. Step 3: The delivery of the  Campylobacter  invasion antigens ( e.g. , CiaD shown in red) to the host cell [  16 ,  19 ,  27 ,  47 ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel  et. al,  Invasion of epithelial cells by  Campylobacter jejuni  is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [  27 ,  28 ,  45 ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in  C. jejuni  internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the  C. jejuni  invasion complex are shown in green.
    Figure Legend Snippet: Model of C. jejuni internalization. C. jejuni invasion of host cells. Step 1: C. jejuni binds to fibronectin (Fn) via the two C. jejuni Fn binding proteins CadF (blue dots) and FlpA (yellow dots) [ 43 , 44 ] causing activation of the α 5 β 1 integrin receptors and the epidermal growth factor receptor (EGFR) [ 27 , 45 ]. Step 2: Activation of the α 5 β 1 integrin leads to the recruitment and partial activation of FAK and paxillin [ 27 , 46 ]. Step 3: The delivery of the Campylobacter invasion antigens ( e.g. , CiaD shown in red) to the host cell [ 16 , 19 , 27 , 47 ] leads to the maximal activation of key components of the focal complex ( i.e. , FAK, paxillin, vinculin, p130Cas, Src, and the CrkII/DOCK-180/ELMO complex) [27,28,45, Konkel et. al, Invasion of epithelial cells by Campylobacter jejuni is independent of caveolin-1, In Submission]. Step 4: Focal complex activation, in conjunction with CiaD, leads to the phosphorylation of Erk 1/2. Caveolin-1, Vav2, Rac1, and Cdc42 are also activated following focal complex activation [ 27 , 28 , 45 ]. Step 5: Activation of Erk 1/2 and Src leads to the phosphorylation of cortactin, which allows for the Rho GTPases Rac1 and Cdc42 to activate N-WASP associated with phosphorylated cortactin, promoting actin cytoskeletal reorganization. Highlighted in this model is the role of CiaD in C. jejuni internalization. Specifically, CiaD is necessary for the maximal activation of the Erk 1/2 and cortactin signaling pathways. Components of the focal complex and focal complex associated proteins are shown in blue. The newly identified components of the C. jejuni invasion complex are shown in green.

    Techniques Used: Binding Assay, Activation Assay

    CiaD is required for Erk 1/2-cortactin association.  INT 407 cells were infected with a  C. jejuni  wild-type strain,  ciaD  mutant,  ciaD  complemented isolate, or uninfected (control) for 45 min.  A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies.  B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P
    Figure Legend Snippet: CiaD is required for Erk 1/2-cortactin association. INT 407 cells were infected with a C. jejuni wild-type strain, ciaD mutant, ciaD complemented isolate, or uninfected (control) for 45 min. A . The cell lysates were subjected to immunoprecipitation experiments with an antibody against cortactin, separated by SDS-PAGE and blotted for cortactin, p-cortactin, N-WASP, and pErk 1/2. Whole cell lysates (WCL) were also probed with an α-cortactin antibody to confirm similar inputs. Also shown are the blots of the IgG isotype control IP probed with cortactin, p-cortactin, N-WASP and pErk 1/2 antibodies. B . Band intensity of p-cortactin, N-WASP, and pErk 1/2 were normalized to total cortactin from three independent experiments. The asterisks indicate a significant difference ( P

    Techniques Used: Infection, Mutagenesis, Immunoprecipitation, SDS Page

    CiaD induced host cell membrane ruffling is Rho GTPase independent. A . Representative scanning electron microscopy images of INT 407 cells; uninfected (Panel 1), infected with a  C. jejuni  wild-type strain (2 and 6),  ciaD  mutant (3 and 7),  ciaD  complemented isolate (4 and 8), and cells infected with a  C. jejuni  wild-type strain that had been pretreated with PD98059, an inhibitor of Erk 1/2 activation (5 and 9). Arrows in higher magnification images show bacteria in direct contact with host cells (6, 7, 8, and 9). INT 407 cells infected with the  C. jejuni  wild-type strain or the  ciaD  complemented isolate show extensive membrane ruffling (6 and 8), and INT 407 cells infected with the  ciaD  mutant or cells infected with a  C. jejuni  wild-type strain that had been pretreated with PD98059 display little host cell membrane ruffling (7 and 9). Images are shown at a magnification of 7,000× with a 10 μM scale bar (1–5), and 50,000× with a 2 μM scale bar (6–9). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. Indicated within each panel is the percent of host cell that display membrane ruffling . B . Rac1 activation in host cells infected with  C. jejuni . Whole cell lysates were processed after 15 min of infection and analyzed for activated Rac1 by G-LISA™. The mean ± SEM of total active Rac1 is indicated in relative optical density.  C . Cdc42 activation in host cells infected with the various  C. jejuni  strains was assessed by G-LISA™. The mean ± SEM of total active Cdc42 is indicated in relative optical density. The asterisks indicate a significant difference ( P
    Figure Legend Snippet: CiaD induced host cell membrane ruffling is Rho GTPase independent. A . Representative scanning electron microscopy images of INT 407 cells; uninfected (Panel 1), infected with a C. jejuni wild-type strain (2 and 6), ciaD mutant (3 and 7), ciaD complemented isolate (4 and 8), and cells infected with a C. jejuni wild-type strain that had been pretreated with PD98059, an inhibitor of Erk 1/2 activation (5 and 9). Arrows in higher magnification images show bacteria in direct contact with host cells (6, 7, 8, and 9). INT 407 cells infected with the C. jejuni wild-type strain or the ciaD complemented isolate show extensive membrane ruffling (6 and 8), and INT 407 cells infected with the ciaD mutant or cells infected with a C. jejuni wild-type strain that had been pretreated with PD98059 display little host cell membrane ruffling (7 and 9). Images are shown at a magnification of 7,000× with a 10 μM scale bar (1–5), and 50,000× with a 2 μM scale bar (6–9). Boxes indicate the area of the INT 407 cell that is shown in the 50,000× panel. Indicated within each panel is the percent of host cell that display membrane ruffling . B . Rac1 activation in host cells infected with C. jejuni . Whole cell lysates were processed after 15 min of infection and analyzed for activated Rac1 by G-LISA™. The mean ± SEM of total active Rac1 is indicated in relative optical density. C . Cdc42 activation in host cells infected with the various C. jejuni strains was assessed by G-LISA™. The mean ± SEM of total active Cdc42 is indicated in relative optical density. The asterisks indicate a significant difference ( P

    Techniques Used: Electron Microscopy, Infection, Mutagenesis, Activation Assay

    Erk 1/2 is necessary for cytosolic signaling required for maximal  C. jejuni  invasion of host cell. A . INT 407 cells were infected with  C. jejuni  incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a  C. jejuni  wild-type strain.  B . Transcription is not required for  C. jejuni  invasion. INT 407 cells were infected with  C. jejuni  and invasion was assessed.  C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various  C. jejuni  strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample.  D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a  C. jejuni  wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P
    Figure Legend Snippet: Erk 1/2 is necessary for cytosolic signaling required for maximal C. jejuni invasion of host cell. A . INT 407 cells were infected with C. jejuni incubated for 6 h, and IL-8 quantified using an IL-8 ELISA. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was added to INT 407 cells for 30 min prior to infection with a C. jejuni wild-type strain. B . Transcription is not required for C. jejuni invasion. INT 407 cells were infected with C. jejuni and invasion was assessed. C . CiaD is required for serine phosphorylation of cortactin. INT 407 cells were infected with the various C. jejuni strains and cellular lysates were prepared. Blots were probed with phospho-specific antibodies to cortactin. The blot was stripped and re-probed with an α-cortactin antibody. Densitometry of p-cortactin is shown as the ratio of p-cortactin to total cortactin (t-cortactin) for each sample. D . Erk 1/2 is required for serine phosphorylation of cortactin. INT 407 cells were pre-treated with PD98059, an inhibitor of Erk 1/2 activation, and infected with a C. jejuni wild-type strain. Blots were probed with a phospho-specific antibody to cortactin. The blot was stripped and re-probed with an α-tubulin antibody. Molecular masses, in kilodaltons (kDa), are indicated on the left. The asterisks indicate a significant difference ( P

    Techniques Used: Infection, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay

    CiaD and Erk 1/2 are required for maximal  C. jejuni  invasion. A . The  C. jejuni ciaD  mutant is deficient in maximal invasion of INT 407 cells. Invasion of INT 407 cells was assessed using a gentamicin protection assay as outlined in Methods.  B . Maximal activation of Erk 1/2 requires CiaD .  INT 407 cells were infected with the various isolates of  C. jejuni  for 45 min and cell lysates were evaluated via immunoblot. Blots were probed with phospho-specific antibodies to Erk 1/2. All blots were stripped and re-probed with an α-Erk 1/2 antibody. Molecular mass size standards, in kilodaltons (kDa), are indicated on the left. Densitometry of pErk 1/2 is shown as the ratio of p-Erk 1/2 to t-Erk 1/2 for each sample.  C . Erk 1/2 kinase activation is required for maximal bacterial invasion. PD98059, an inhibitor of Erk 1/2 activation, was added to INT 407 cells for 30 min prior to inoculation with a  C. jejuni  wild-type strain, and bacterial invasion was assessed. The asterisk indicates a significant reduction compared to the value obtained for the  C. jejuni  wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis ( P
    Figure Legend Snippet: CiaD and Erk 1/2 are required for maximal C. jejuni invasion. A . The C. jejuni ciaD mutant is deficient in maximal invasion of INT 407 cells. Invasion of INT 407 cells was assessed using a gentamicin protection assay as outlined in Methods. B . Maximal activation of Erk 1/2 requires CiaD . INT 407 cells were infected with the various isolates of C. jejuni for 45 min and cell lysates were evaluated via immunoblot. Blots were probed with phospho-specific antibodies to Erk 1/2. All blots were stripped and re-probed with an α-Erk 1/2 antibody. Molecular mass size standards, in kilodaltons (kDa), are indicated on the left. Densitometry of pErk 1/2 is shown as the ratio of p-Erk 1/2 to t-Erk 1/2 for each sample. C . Erk 1/2 kinase activation is required for maximal bacterial invasion. PD98059, an inhibitor of Erk 1/2 activation, was added to INT 407 cells for 30 min prior to inoculation with a C. jejuni wild-type strain, and bacterial invasion was assessed. The asterisk indicates a significant reduction compared to the value obtained for the C. jejuni wild-type strain, as judged by one-way ANOVA followed by post-hoc Dunnett’s analysis ( P

    Techniques Used: Mutagenesis, Activation Assay, Infection

    12) Product Images from "Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis"

    Article Title: Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191696

    iAβ expression leads to diminished EGF-dependent ERK1/2 phosphorylation. (A)  Immunoblot of phospho-ERK 1/2 and total-ERK 1/2 in VC or iAβ transduced HEK293 cells following EGF stimulation. While total-ERK 1/2 remains unaltered, decreased EGF dependent phospho-ERK 1/2 levels observed in iAβ-transduced cells relative to VC. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control.  (B)  Plot of the relative densitometry analysis in biological replicates depicting a relative decrease in phospho-ERK 1/2 levels with data shown as mean ± SE (n = 3) with statistical significance (**p
    Figure Legend Snippet: iAβ expression leads to diminished EGF-dependent ERK1/2 phosphorylation. (A) Immunoblot of phospho-ERK 1/2 and total-ERK 1/2 in VC or iAβ transduced HEK293 cells following EGF stimulation. While total-ERK 1/2 remains unaltered, decreased EGF dependent phospho-ERK 1/2 levels observed in iAβ-transduced cells relative to VC. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control. (B) Plot of the relative densitometry analysis in biological replicates depicting a relative decrease in phospho-ERK 1/2 levels with data shown as mean ± SE (n = 3) with statistical significance (**p

    Techniques Used: Expressing, Two-Dimensional Gel Electrophoresis, Transformation Assay

    Differential influence of eAβ and iAβ on MAPK activity. (A)  Confirmation of successful generation of extracellular monomeric, oligomer and fibrillar quaternary organization. Coomassie brilliant blue stained, 15% SDS PAGE gel, showing the varying eAβ quaternary organization states at 4°C and 37°C oligomerization protocols. Preferential staining of lower order eAβ organization observed using coomassie staining at 4°C. Sypro  ®  Ruby stained, 15% SDS PAGE gel, of eAβ 1–42  revealing the higher order quaternary organization states.  (B)  Immunoblot of the phospho-ERK 1/2 and total-ERK 1/2 in VC or iAβ transduced HEK293 cells following 24 h pretreatment with monomeric/oligomeric/fibrillary (M/O/F) eAβ and subsequent EGF stimulation. No differences in EGF responsiveness observed in the DMSO or M/O/F treated VC samples. Decreased ERK 1/2 phosphorylation seen in iAβ transduced cells treated with M/O/F relative iAβ transduced cells treated with DMSO alone. Beta-actin employed as loading control.  (C)  Plot of the relative densitometry analysis of phospho-ERK 1/2 intensity in VC or iAβ after treatment with DMSO or M/O/F of biological replicates with data shown as mean ± SE (n = 3) and statistical significance.  (D)  Plot of total-ERK levels demonstrate intensity remains unchanged in all treatment categories. Complete pairwise analysis shown in   Table 1 .
    Figure Legend Snippet: Differential influence of eAβ and iAβ on MAPK activity. (A) Confirmation of successful generation of extracellular monomeric, oligomer and fibrillar quaternary organization. Coomassie brilliant blue stained, 15% SDS PAGE gel, showing the varying eAβ quaternary organization states at 4°C and 37°C oligomerization protocols. Preferential staining of lower order eAβ organization observed using coomassie staining at 4°C. Sypro ® Ruby stained, 15% SDS PAGE gel, of eAβ 1–42 revealing the higher order quaternary organization states. (B) Immunoblot of the phospho-ERK 1/2 and total-ERK 1/2 in VC or iAβ transduced HEK293 cells following 24 h pretreatment with monomeric/oligomeric/fibrillary (M/O/F) eAβ and subsequent EGF stimulation. No differences in EGF responsiveness observed in the DMSO or M/O/F treated VC samples. Decreased ERK 1/2 phosphorylation seen in iAβ transduced cells treated with M/O/F relative iAβ transduced cells treated with DMSO alone. Beta-actin employed as loading control. (C) Plot of the relative densitometry analysis of phospho-ERK 1/2 intensity in VC or iAβ after treatment with DMSO or M/O/F of biological replicates with data shown as mean ± SE (n = 3) and statistical significance. (D) Plot of total-ERK levels demonstrate intensity remains unchanged in all treatment categories. Complete pairwise analysis shown in Table 1 .

    Techniques Used: Activity Assay, Staining, SDS Page

    Altered ERK 1/2 and Smad 1/5/8 phosphorylation observed in primary rat hippocampal cells upon iAβ expression. (A)  Immunoblot of phospho-ERK 1/2, total-ERK 1/2, and MYC-Tag in VC or iAβ transduced HPC neurons following BDNF stimulation. Myc-tag observed in lanes corresponding to iAβ transduced hippocampal cells and absent in VC samples. Decreased ERK 1/2 phosphorylation in iAβ expressing cells is rescued upon pre-treatment with PLX4032. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control.  (B)  Plot of relative densitometry analysis of phospho-ERK 1/2 with data corresponding to biological replicates shown as mean ± SE (n = 3). Statistically significant (p
    Figure Legend Snippet: Altered ERK 1/2 and Smad 1/5/8 phosphorylation observed in primary rat hippocampal cells upon iAβ expression. (A) Immunoblot of phospho-ERK 1/2, total-ERK 1/2, and MYC-Tag in VC or iAβ transduced HPC neurons following BDNF stimulation. Myc-tag observed in lanes corresponding to iAβ transduced hippocampal cells and absent in VC samples. Decreased ERK 1/2 phosphorylation in iAβ expressing cells is rescued upon pre-treatment with PLX4032. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control. (B) Plot of relative densitometry analysis of phospho-ERK 1/2 with data corresponding to biological replicates shown as mean ± SE (n = 3). Statistically significant (p

    Techniques Used: Expressing, Two-Dimensional Gel Electrophoresis, Transformation Assay

    Characterization of primary rat hippocampal (HPC) and cortical (CTX) neuronal sensitivity to growth factor and cytokine. (A)  Immunoblot of phospho-ERK 1/2 and total-ERK1/2 in DIV21 primary HPC and CTX cells following treatment with DMSO, NGF alone, or PLX4032 pre-treatment and subsequent NGF stimulation. Beta-actin employed as loading control. Although total-ERK 1/2 remains consistent among the treatment groups in both HPC and CTX cells, ERK 1/2 phosphorylation is observed most prominently in HPC cells.  (B)  Plot of the relative phospho-ERK 1/2 abundance relative to DMSO treated HPC cells illustrates the distinct sensitivities of the HPC and CTX populations. Pre-treatment with PLX4032 leads to modest, but statistically significant, increase in NGF dependent ERK 1/2 phosphorylation. Complete pairwise analysis shown in   Table 2 .  (C)  Immunoblot of phosphorylated and total ERK 1/2 in DIV21 primary HPC and CTX cells following treatment with DMSO, PLX4032, EGF, or BDNF. Beta-actin employed as loading control. Increased HPC sensitivity, relative to CTX cells, can be observed across all treatment combinations.  (D)  Densitometry analysis of relative phospho-ERK 1/2 abundance illustrating the increased HPC to CTX cell sensitivity. Complete pairwise analysis shown in   Table 3 .  (E)  Immunoblot of phospho-Smad 1/5/8 and total-Smad 1/5/8 of DIV21 primary HPC and CTX cells following treatment with rh-BMP2 alone or in combination with the BMP-2 receptor inhibitor Dorsomorphin. Beta-Actin employed as loading control. Increased hippocampal responsiveness observed relative to cortical samples.  (F)  Plot of densitometry analysis illustrating the increased HPC sensitivity relative to CTX cells. Complete pairwise analysis shown in   Table 4 .
    Figure Legend Snippet: Characterization of primary rat hippocampal (HPC) and cortical (CTX) neuronal sensitivity to growth factor and cytokine. (A) Immunoblot of phospho-ERK 1/2 and total-ERK1/2 in DIV21 primary HPC and CTX cells following treatment with DMSO, NGF alone, or PLX4032 pre-treatment and subsequent NGF stimulation. Beta-actin employed as loading control. Although total-ERK 1/2 remains consistent among the treatment groups in both HPC and CTX cells, ERK 1/2 phosphorylation is observed most prominently in HPC cells. (B) Plot of the relative phospho-ERK 1/2 abundance relative to DMSO treated HPC cells illustrates the distinct sensitivities of the HPC and CTX populations. Pre-treatment with PLX4032 leads to modest, but statistically significant, increase in NGF dependent ERK 1/2 phosphorylation. Complete pairwise analysis shown in Table 2 . (C) Immunoblot of phosphorylated and total ERK 1/2 in DIV21 primary HPC and CTX cells following treatment with DMSO, PLX4032, EGF, or BDNF. Beta-actin employed as loading control. Increased HPC sensitivity, relative to CTX cells, can be observed across all treatment combinations. (D) Densitometry analysis of relative phospho-ERK 1/2 abundance illustrating the increased HPC to CTX cell sensitivity. Complete pairwise analysis shown in Table 3 . (E) Immunoblot of phospho-Smad 1/5/8 and total-Smad 1/5/8 of DIV21 primary HPC and CTX cells following treatment with rh-BMP2 alone or in combination with the BMP-2 receptor inhibitor Dorsomorphin. Beta-Actin employed as loading control. Increased hippocampal responsiveness observed relative to cortical samples. (F) Plot of densitometry analysis illustrating the increased HPC sensitivity relative to CTX cells. Complete pairwise analysis shown in Table 4 .

    Techniques Used:

    PLX4032 rescue iAβ mediated attenuation of ERK 1/2 phosphorylation. (A)  Immunoblot for phospho-ERK 1/2, total-ERK 1/2, and phospho-c-RAF (Ser 259) in VC or iAβ transduced HEK293 cells following pretreatment with PLX4032 (30 min) and subsequent vehicle or EGF stimulation. Loss of ERK 1/2 phosphorylation is rescued upon pre-treatment with PLX4032. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control.  (B)  Plot of the relative densitometry analysis of phospho-ERK 1/2 intensity of biological replicates with data shown as mean ± SE (n = 3).  (C)  Measurement of total-ERK 1/2 intensity demonstrate unaffected levels following treatment in VC or iAβ transduced cells.  (D)  Histogram of the phospho-flow cytometry measurement of EGF treated VC (gray) or iAβ (black) transduced HEK293 cells for ERK 1/2 phosphorylation. Positive shift in the MFI of iAβ transduced cells reflects an EGF dependent and PLX4032 mediated increase in ERK 1/2 phosphorylation.  (E)  Replicate analysis shown as mean ± SE (n = 3) of the MFI with a statistically significan t (*p
    Figure Legend Snippet: PLX4032 rescue iAβ mediated attenuation of ERK 1/2 phosphorylation. (A) Immunoblot for phospho-ERK 1/2, total-ERK 1/2, and phospho-c-RAF (Ser 259) in VC or iAβ transduced HEK293 cells following pretreatment with PLX4032 (30 min) and subsequent vehicle or EGF stimulation. Loss of ERK 1/2 phosphorylation is rescued upon pre-treatment with PLX4032. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control. (B) Plot of the relative densitometry analysis of phospho-ERK 1/2 intensity of biological replicates with data shown as mean ± SE (n = 3). (C) Measurement of total-ERK 1/2 intensity demonstrate unaffected levels following treatment in VC or iAβ transduced cells. (D) Histogram of the phospho-flow cytometry measurement of EGF treated VC (gray) or iAβ (black) transduced HEK293 cells for ERK 1/2 phosphorylation. Positive shift in the MFI of iAβ transduced cells reflects an EGF dependent and PLX4032 mediated increase in ERK 1/2 phosphorylation. (E) Replicate analysis shown as mean ± SE (n = 3) of the MFI with a statistically significan t (*p

    Techniques Used: Two-Dimensional Gel Electrophoresis, Transformation Assay, Flow Cytometry, Cytometry

    13) Product Images from "Caveolin-1 Deficiency (-/-) Conveys Premalignant Alterations in Mammary Epithelia, with Abnormal Lumen Formation, Growth Factor Independence, and Cell Invasiveness"

    Article Title: Caveolin-1 Deficiency (-/-) Conveys Premalignant Alterations in Mammary Epithelia, with Abnormal Lumen Formation, Growth Factor Independence, and Cell Invasiveness

    Journal: The American Journal of Pathology

    doi:

    Cav-1-null acini show hyperactivation of the p42/44-MAP kinase signaling cascade, and EGF-independent growth. A: Hyperactivation of the Ras-p42/44-MAP kinase pathway in Cav-1 KO acini. Lysates from day 18 WT and Cav-1-deficient acini were subjected to Western blot analysis with antibodies against the activated phosphorylated form of ERK-1/2. Note that the Ras-p42/44-MAP kinase signaling cascade is hyperactivated in Cav-1-null acini. Equal loading was assessed by Western blot with a control phospho-independent antibody that recognizes total ERK-1/2. Immunoblotting with E-cadherin is shown as an additional control for equal epithelial protein content. B–D: EGF-independent growth of Cav-1-null acini. To evaluate whether loss of Cav-1 imparts growth factor independence, WT and Cav-1 KO three-dimensional epithelial structures were cultured either in the absence or in the presence of EGF, for up to 12 days. Images were acquired at days 4, 8, and 12. B: Phase images of EGF-deprived acini. In the absence of EGF, WT acini are very small and unable to grow. On the contrary, Cav-1-null mammary epithelial cells retain the ability to proliferate and to form acini-like spheroids without EGF. C: Acinar growth: size. The diameter of WT and Cav-1 KO acini, cultured with or without EGF, was measured at days 4, 8, and 12. At least 50 acini for each genotype were scored at any given time point. As expected, in the presence of EGF, Cav-1-null acini display an obvious delay in reaching a growth-arrested state (day 12 versus day 8 of WT acini), and exhibit an approximately twofold size increase, as compared to WT acini. However, in the absence of EGF, WT mammary epithelial cells develop as very small spheroids, which do not enlarge, suggesting that EGF is normally required for the growth and proliferation of three-dimensional cultures of mammary epithelial cells. In striking contrast, Cav-1-deficient acini cultured without EGF grow at a comparable rate as when they are cultured in the presence of EGF. More than 50 acini for each genotype were scored at a given time point. * P ≤ 0.00004. Error bars, SEM. D: Acinar growth: number of acini per high-power field. WT and Cav-1 KO acini were cultured with or without EGF in parallel experiments. The number of acini per high-power field was counted at days 4, 8, and 12. At least 20 high-power fields were scored for each genotype at any given time point. Note that, when cultured with EGF, WT and Cav-1 KO mammary epithelial cells form a similar number of acini, demonstrating that we seeded a comparable amount of WT and Cav-1-null cells into the wells. However, parallel experiments performed in the absence of EGF, show that the number of WT, but not that of Cav-1 KO acini, is greatly affected by a lack of EGF. Remarkably, in the absence of EGF, the number of Cav-1 KO acini is approximately fourfold increased, as compared to WT acini. * P ≤ 0.00018. Error bars, SEM.
    Figure Legend Snippet: Cav-1-null acini show hyperactivation of the p42/44-MAP kinase signaling cascade, and EGF-independent growth. A: Hyperactivation of the Ras-p42/44-MAP kinase pathway in Cav-1 KO acini. Lysates from day 18 WT and Cav-1-deficient acini were subjected to Western blot analysis with antibodies against the activated phosphorylated form of ERK-1/2. Note that the Ras-p42/44-MAP kinase signaling cascade is hyperactivated in Cav-1-null acini. Equal loading was assessed by Western blot with a control phospho-independent antibody that recognizes total ERK-1/2. Immunoblotting with E-cadherin is shown as an additional control for equal epithelial protein content. B–D: EGF-independent growth of Cav-1-null acini. To evaluate whether loss of Cav-1 imparts growth factor independence, WT and Cav-1 KO three-dimensional epithelial structures were cultured either in the absence or in the presence of EGF, for up to 12 days. Images were acquired at days 4, 8, and 12. B: Phase images of EGF-deprived acini. In the absence of EGF, WT acini are very small and unable to grow. On the contrary, Cav-1-null mammary epithelial cells retain the ability to proliferate and to form acini-like spheroids without EGF. C: Acinar growth: size. The diameter of WT and Cav-1 KO acini, cultured with or without EGF, was measured at days 4, 8, and 12. At least 50 acini for each genotype were scored at any given time point. As expected, in the presence of EGF, Cav-1-null acini display an obvious delay in reaching a growth-arrested state (day 12 versus day 8 of WT acini), and exhibit an approximately twofold size increase, as compared to WT acini. However, in the absence of EGF, WT mammary epithelial cells develop as very small spheroids, which do not enlarge, suggesting that EGF is normally required for the growth and proliferation of three-dimensional cultures of mammary epithelial cells. In striking contrast, Cav-1-deficient acini cultured without EGF grow at a comparable rate as when they are cultured in the presence of EGF. More than 50 acini for each genotype were scored at a given time point. * P ≤ 0.00004. Error bars, SEM. D: Acinar growth: number of acini per high-power field. WT and Cav-1 KO acini were cultured with or without EGF in parallel experiments. The number of acini per high-power field was counted at days 4, 8, and 12. At least 20 high-power fields were scored for each genotype at any given time point. Note that, when cultured with EGF, WT and Cav-1 KO mammary epithelial cells form a similar number of acini, demonstrating that we seeded a comparable amount of WT and Cav-1-null cells into the wells. However, parallel experiments performed in the absence of EGF, show that the number of WT, but not that of Cav-1 KO acini, is greatly affected by a lack of EGF. Remarkably, in the absence of EGF, the number of Cav-1 KO acini is approximately fourfold increased, as compared to WT acini. * P ≤ 0.00018. Error bars, SEM.

    Techniques Used: Western Blot, Cell Culture

    14) Product Images from "A Pan-GTPase Inhibitor as a Molecular Probe"

    Article Title: A Pan-GTPase Inhibitor as a Molecular Probe

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134317

    Effects of compound 1 on the Ras-dependent H358 cells. The U937 FPRΔST cell line was used as control since it had no known Ras-dependency. (A) Compound  1  decreased the viability of H358 cells at 48 h, compared to the control. (B) Apoptosis analysis showed increase response of H358 cells over time compared to control cells. (C) Treatment of compound  1  decreased the phosphorylation level of ERK 1/2 in H358 cells. (-) 0.1% DMSO treated controls; (+) 10 μM compound  1  treated samples. Cells were either grown in complete medium (lane 1 and 2) or stimulated after starvation (lane 3 and 4). The experiment was conducted three times. A representative immunoblot is shown. Immunoblots from all three experiments were quantified by densitometry using exposures in the linear range. The relative ratios of phospho Erk1/2 to total Erk1/2 are plotted.  p  = 0.0142, calculated with an unpaired two-tailed t-test using GraphPad Prism.
    Figure Legend Snippet: Effects of compound 1 on the Ras-dependent H358 cells. The U937 FPRΔST cell line was used as control since it had no known Ras-dependency. (A) Compound 1 decreased the viability of H358 cells at 48 h, compared to the control. (B) Apoptosis analysis showed increase response of H358 cells over time compared to control cells. (C) Treatment of compound 1 decreased the phosphorylation level of ERK 1/2 in H358 cells. (-) 0.1% DMSO treated controls; (+) 10 μM compound 1 treated samples. Cells were either grown in complete medium (lane 1 and 2) or stimulated after starvation (lane 3 and 4). The experiment was conducted three times. A representative immunoblot is shown. Immunoblots from all three experiments were quantified by densitometry using exposures in the linear range. The relative ratios of phospho Erk1/2 to total Erk1/2 are plotted. p = 0.0142, calculated with an unpaired two-tailed t-test using GraphPad Prism.

    Techniques Used: Western Blot, Two Tailed Test

    15) Product Images from "ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury"

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    Journal: Neurochemical Research

    doi: 10.1007/s11064-018-2558-z

    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition. a and b Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Techniques Used: Activation Assay, Activity Assay, Inhibition, Western Blot, Cell Culture

    The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery
    Figure Legend Snippet: The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Techniques Used: Activity Assay, Functional Assay

    BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals.  a  Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126.  b  Quantification analysis of the infarct volume [n = 6, *P 
    Figure Legend Snippet: BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals. a Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126. b Quantification analysis of the infarct volume [n = 6, *P 

    Techniques Used: Inhibition, Activation Assay, Staining

    BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation.  a  Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 
    Figure Legend Snippet: BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation. a Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Techniques Used: Functional Assay, Inhibition, Activation Assay, Injection

    After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased.  a  and  b  Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm.  c  and  d  Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 
    Figure Legend Snippet: After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased. a and b Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm. c and d Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Techniques Used: Double Immunofluorescence Staining, Western Blot, Expressing

    BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury.  a  A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure.  b  and  c  Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 
    Figure Legend Snippet: BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury. a A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure. b and c Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Techniques Used: Western Blot, Expressing

    BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level.  a  Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level. a Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Techniques Used: Activity Assay, Western Blot, Expressing

    16) Product Images from "Renalase attenuates hypertension, renal injury and cardiac remodelling in rats with subtotal nephrectomy"

    Article Title: Renalase attenuates hypertension, renal injury and cardiac remodelling in rats with subtotal nephrectomy

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12813

    Ad‐renalase treatment normalized cardiac expression of pro‐fibrotic markers and phosphorylated  ERK ‐1/2 in  CKD  rats. Representative Western blot and quantification of  TGF ‐β ( A ),  MMP ‐1 ( B ),  TIMP ‐1 ( C ) and phosphorylated  ERK ‐1/2 ( D ) expression in left ventricle from three groups at week 6. All values are presented as means ± S.E.M. ( n  = 10). * P
    Figure Legend Snippet: Ad‐renalase treatment normalized cardiac expression of pro‐fibrotic markers and phosphorylated ERK ‐1/2 in CKD rats. Representative Western blot and quantification of TGF ‐β ( A ), MMP ‐1 ( B ), TIMP ‐1 ( C ) and phosphorylated ERK ‐1/2 ( D ) expression in left ventricle from three groups at week 6. All values are presented as means ± S.E.M. ( n = 10). * P

    Techniques Used: Expressing, Western Blot

    Ad‐renalase treatment suppressed  ERK ‐1/2 phosphorylation in the remnant kidney. Representative Western blots of phosphorylated  ERK ‐1/2 ( A ) and phosphorylated p38 ( B ) in kidney. Values are presented as means ± S.E.M. ( n  = 10). * P
    Figure Legend Snippet: Ad‐renalase treatment suppressed ERK ‐1/2 phosphorylation in the remnant kidney. Representative Western blots of phosphorylated ERK ‐1/2 ( A ) and phosphorylated p38 ( B ) in kidney. Values are presented as means ± S.E.M. ( n = 10). * P

    Techniques Used: Western Blot

    17) Product Images from "Interleukin-6 in the bone marrow microenvironment promotes the growth and survival of neuroblastoma cells"

    Article Title: Interleukin-6 in the bone marrow microenvironment promotes the growth and survival of neuroblastoma cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-08-0613

    IL-6 activates STAT-3 and Erk 1/2 in IL-6R positive CHLA-255 neuroblastoma cells.  A,  expression of phospho-STAT-3 (pSTAT-3), STAT-3, phospho-Erk 1/2 (pErk 1/2) and Erk 1/2 examined by Western blot analysis in total cell lysates (20 μg) of CHLA-255
    Figure Legend Snippet: IL-6 activates STAT-3 and Erk 1/2 in IL-6R positive CHLA-255 neuroblastoma cells. A, expression of phospho-STAT-3 (pSTAT-3), STAT-3, phospho-Erk 1/2 (pErk 1/2) and Erk 1/2 examined by Western blot analysis in total cell lysates (20 μg) of CHLA-255

    Techniques Used: Expressing, Western Blot

    18) Product Images from "DISC1 Regulates Neurotrophin-Induced Axon Elongation via Interaction with Grb2"

    Article Title: DISC1 Regulates Neurotrophin-Induced Axon Elongation via Interaction with Grb2

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3825-06.2007

    Requirement of DISC1 in the phosphorylation of ERK-1/2. A , Hippocampal neurons were cotransfected with the expression plasmid encoding EGFP and scramble or rDISC1–siRNAs at DIV0. Neurons were cultured for 3 d and then treated with vehicle or NT-3 (100 ng/ml) for 30 min. Cells were fixed and immunostained with antibodies against GFP and phospho-ERK-1/2. Cells were also stained with the cytoplasmic fluorescent probe CMTMR before fixation. Enlarged images of the cell bodies (1) and growth cones (2) of each EGFP-positive neuron are shown. Scale bars, 20 μm. B , Hippocampal neurons were cotransfected with the expression vector encoding EGFP or EGFP–DISC1 and scramble or rDISC1–siRNAs at DIV0. Neurons were cultured for 3 d and then treated with vehicle or NT-3 (100 ng/ml) for 30 min. Cells were fixed and immunostained with antibodies against GFP and phospho-ERK-1/2. The fluorescence intensity of immunostained phospho-ERK-1/2 at cell bodies and growth cones were assessed (see Materials and Methods). Numbers of cells used for each calculation are > 50, and the values shown are means ± SE of three independent experiments (Student’s t test, ** p
    Figure Legend Snippet: Requirement of DISC1 in the phosphorylation of ERK-1/2. A , Hippocampal neurons were cotransfected with the expression plasmid encoding EGFP and scramble or rDISC1–siRNAs at DIV0. Neurons were cultured for 3 d and then treated with vehicle or NT-3 (100 ng/ml) for 30 min. Cells were fixed and immunostained with antibodies against GFP and phospho-ERK-1/2. Cells were also stained with the cytoplasmic fluorescent probe CMTMR before fixation. Enlarged images of the cell bodies (1) and growth cones (2) of each EGFP-positive neuron are shown. Scale bars, 20 μm. B , Hippocampal neurons were cotransfected with the expression vector encoding EGFP or EGFP–DISC1 and scramble or rDISC1–siRNAs at DIV0. Neurons were cultured for 3 d and then treated with vehicle or NT-3 (100 ng/ml) for 30 min. Cells were fixed and immunostained with antibodies against GFP and phospho-ERK-1/2. The fluorescence intensity of immunostained phospho-ERK-1/2 at cell bodies and growth cones were assessed (see Materials and Methods). Numbers of cells used for each calculation are > 50, and the values shown are means ± SE of three independent experiments (Student’s t test, ** p

    Techniques Used: Expressing, Plasmid Preparation, Cell Culture, Staining, Fluorescence

    19) Product Images from "The phospholipase DDHD1 as a new target in colorectal cancer therapy"

    Article Title: The phospholipase DDHD1 as a new target in colorectal cancer therapy

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0753-z

    Effects of DDHD1 silencing on ERK and Akt signaling pathways.  a  Levels of ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 transfected with scrambled or DDHD1 siRNA using the Bio-Plex Pro Cell Signaling Assay (left panel) and western blot (right panel).  b  Levels of Akt and phospho-Akt were determined by the Bio-Plex Multiple Cytokine Assay (upper panel) and by FACS analysis (lower panel) in the SW480 transfected with scrambled or DDHD1 siRNA (* p  ≤ 0.05; ** p  ≤ 0.01)
    Figure Legend Snippet: Effects of DDHD1 silencing on ERK and Akt signaling pathways. a Levels of ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 transfected with scrambled or DDHD1 siRNA using the Bio-Plex Pro Cell Signaling Assay (left panel) and western blot (right panel). b Levels of Akt and phospho-Akt were determined by the Bio-Plex Multiple Cytokine Assay (upper panel) and by FACS analysis (lower panel) in the SW480 transfected with scrambled or DDHD1 siRNA (* p  ≤ 0.05; ** p  ≤ 0.01)

    Techniques Used: Transfection, Western Blot, Cytokine Assay, FACS

    In vitro and in vivo effects of DDHD1 overexpression on colon cancer cell growth.  a  Cell viability was measured by MTT assay on SW480 cell lines after 24, 48, 72 or 96 h of transfection with mock or DDHD1 plasmid DNA. The values were plotted as % of growth vs Ctrl (cells transfected with mock plasmid). Each point represents the mean ± SD of three independent experiments. Asterisks indicate statistically significant values in comparison to control (mock) (**p ≤ 0.01).  b  Levels of DDHD1, ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 cells transfected with mock or DDHD1 plasmid DNA. Blots were probed with an antibody against GAPDH to ensure equal loading.  c  SW480 cells, transfected with mock or DDHD1 plasmid DNA were injected subcutaneously in nude mice as described. Comparison of the mean tumor volume was an index of the pro-tumor effect of DDHD1. A significant interaction was found between (group) and within (experimental time)-subject factors for volumetric data (F = 6.92,  p  = 0.028). Significant differences in terms of tumor volume were observed between the two groups (8 days  p  = 0.009; 12 days  p  = 0.010; 15 days  p  = 0.024) and within the experimental time (mock at 8 days versus starting point  a , p  = 0.025; over DDHD1 at 8 days versus starting point  b , p  = 0.0001; over DDHD1 at 12 days versus 8 days  c , p  = 0.003; over DDHD1 at 15 days versus 12 days  d , p  = 0.002).  d  IL8 and Survivin mRNA levels were evaluated in tumor biopsies by Real-time PCR.  e  Protein levels of ERK 1/2, phospho-ERK 1/2 and SURVIVIN were analyzed in the mice tumor biopsies
    Figure Legend Snippet: In vitro and in vivo effects of DDHD1 overexpression on colon cancer cell growth. a Cell viability was measured by MTT assay on SW480 cell lines after 24, 48, 72 or 96 h of transfection with mock or DDHD1 plasmid DNA. The values were plotted as % of growth vs Ctrl (cells transfected with mock plasmid). Each point represents the mean ± SD of three independent experiments. Asterisks indicate statistically significant values in comparison to control (mock) (**p ≤ 0.01). b Levels of DDHD1, ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 cells transfected with mock or DDHD1 plasmid DNA. Blots were probed with an antibody against GAPDH to ensure equal loading. c SW480 cells, transfected with mock or DDHD1 plasmid DNA were injected subcutaneously in nude mice as described. Comparison of the mean tumor volume was an index of the pro-tumor effect of DDHD1. A significant interaction was found between (group) and within (experimental time)-subject factors for volumetric data (F = 6.92, p  = 0.028). Significant differences in terms of tumor volume were observed between the two groups (8 days p  = 0.009; 12 days p  = 0.010; 15 days p  = 0.024) and within the experimental time (mock at 8 days versus starting point a , p  = 0.025; over DDHD1 at 8 days versus starting point b , p  = 0.0001; over DDHD1 at 12 days versus 8 days c , p  = 0.003; over DDHD1 at 15 days versus 12 days d , p  = 0.002). d IL8 and Survivin mRNA levels were evaluated in tumor biopsies by Real-time PCR. e Protein levels of ERK 1/2, phospho-ERK 1/2 and SURVIVIN were analyzed in the mice tumor biopsies

    Techniques Used: In Vitro, In Vivo, Over Expression, MTT Assay, Transfection, Plasmid Preparation, Injection, Mouse Assay, Real-time Polymerase Chain Reaction

    20) Product Images from "The phospholipase DDHD1 as a new target in colorectal cancer therapy"

    Article Title: The phospholipase DDHD1 as a new target in colorectal cancer therapy

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0753-z

    Effects of DDHD1 silencing on ERK and Akt signaling pathways.  a  Levels of ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 transfected with scrambled or DDHD1 siRNA using the Bio-Plex Pro Cell Signaling Assay (left panel) and western blot (right panel).  b  Levels of Akt and phospho-Akt were determined by the Bio-Plex Multiple Cytokine Assay (upper panel) and by FACS analysis (lower panel) in the SW480 transfected with scrambled or DDHD1 siRNA (* p  ≤ 0.05; ** p  ≤ 0.01)
    Figure Legend Snippet: Effects of DDHD1 silencing on ERK and Akt signaling pathways. a Levels of ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 transfected with scrambled or DDHD1 siRNA using the Bio-Plex Pro Cell Signaling Assay (left panel) and western blot (right panel). b Levels of Akt and phospho-Akt were determined by the Bio-Plex Multiple Cytokine Assay (upper panel) and by FACS analysis (lower panel) in the SW480 transfected with scrambled or DDHD1 siRNA (* p  ≤ 0.05; ** p  ≤ 0.01)

    Techniques Used: Transfection, Western Blot, Cytokine Assay, FACS

    In vitro and in vivo effects of DDHD1 overexpression on colon cancer cell growth.  a  Cell viability was measured by MTT assay on SW480 cell lines after 24, 48, 72 or 96 h of transfection with mock or DDHD1 plasmid DNA. The values were plotted as % of growth vs Ctrl (cells transfected with mock plasmid). Each point represents the mean ± SD of three independent experiments. Asterisks indicate statistically significant values in comparison to control (mock) (**p ≤ 0.01).  b  Levels of DDHD1, ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 cells transfected with mock or DDHD1 plasmid DNA. Blots were probed with an antibody against GAPDH to ensure equal loading.  c  SW480 cells, transfected with mock or DDHD1 plasmid DNA were injected subcutaneously in nude mice as described. Comparison of the mean tumor volume was an index of the pro-tumor effect of DDHD1. A significant interaction was found between (group) and within (experimental time)-subject factors for volumetric data (F = 6.92,  p  = 0.028). Significant differences in terms of tumor volume were observed between the two groups (8 days  p  = 0.009; 12 days  p  = 0.010; 15 days  p  = 0.024) and within the experimental time (mock at 8 days versus starting point  a , p  = 0.025; over DDHD1 at 8 days versus starting point  b , p  = 0.0001; over DDHD1 at 12 days versus 8 days  c , p  = 0.003; over DDHD1 at 15 days versus 12 days  d , p  = 0.002).  d  IL8 and Survivin mRNA levels were evaluated in tumor biopsies by Real-time PCR.  e  Protein levels of ERK 1/2, phospho-ERK 1/2 and SURVIVIN were analyzed in the mice tumor biopsies
    Figure Legend Snippet: In vitro and in vivo effects of DDHD1 overexpression on colon cancer cell growth. a Cell viability was measured by MTT assay on SW480 cell lines after 24, 48, 72 or 96 h of transfection with mock or DDHD1 plasmid DNA. The values were plotted as % of growth vs Ctrl (cells transfected with mock plasmid). Each point represents the mean ± SD of three independent experiments. Asterisks indicate statistically significant values in comparison to control (mock) (**p ≤ 0.01). b Levels of DDHD1, ERK 1/2, phospho-ERK 1/2 were determined in the cell lysate of SW480 cells transfected with mock or DDHD1 plasmid DNA. Blots were probed with an antibody against GAPDH to ensure equal loading. c SW480 cells, transfected with mock or DDHD1 plasmid DNA were injected subcutaneously in nude mice as described. Comparison of the mean tumor volume was an index of the pro-tumor effect of DDHD1. A significant interaction was found between (group) and within (experimental time)-subject factors for volumetric data (F = 6.92, p  = 0.028). Significant differences in terms of tumor volume were observed between the two groups (8 days p  = 0.009; 12 days p  = 0.010; 15 days p  = 0.024) and within the experimental time (mock at 8 days versus starting point a , p  = 0.025; over DDHD1 at 8 days versus starting point b , p  = 0.0001; over DDHD1 at 12 days versus 8 days c , p  = 0.003; over DDHD1 at 15 days versus 12 days d , p  = 0.002). d IL8 and Survivin mRNA levels were evaluated in tumor biopsies by Real-time PCR. e Protein levels of ERK 1/2, phospho-ERK 1/2 and SURVIVIN were analyzed in the mice tumor biopsies

    Techniques Used: In Vitro, In Vivo, Over Expression, MTT Assay, Transfection, Plasmid Preparation, Injection, Mouse Assay, Real-time Polymerase Chain Reaction

    21) Product Images from "Identification of multiple MAPK-mediated transcription factors regulated by tobacco smoke in airway epithelial cells"

    Article Title: Identification of multiple MAPK-mediated transcription factors regulated by tobacco smoke in airway epithelial cells

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00345.2006

    Decrease of TS-induced NF-κB binding and promoter activity by ERK 1/2 inhibitors.  A  and  B : extracted nuclear proteins without or with TS exposure were added into 96-well plates that had prebound NF-κB duplex DNA. Microwells bearing the biotinylated NF-κB DNA probe (1 pmol) were incubated with an increasing excess of the nonbiotinylated probe, containing either the wild-type (wt) or the mutated NF-κB-binding consensus sequence. Specific DNA binding activity for either p65 ( A ) or p50 ( B ) was determined by measured activity after incubation with respective primary antibody against p65 or p50.  C : A549 cells were transiently transfected with a NF-κB-containing plasmid linked to the pGL3 basic vector and then treated without or with ERK 1/2 inhibitors. After TS exposure, cell supernatants were collected and assayed for luciferase activity as described in MATERIALS AND METHODS. Triplicate samples were harvested from each condition, and results are expressed as relative fold increase in luciferase activity compared with empty vector control. * P
    Figure Legend Snippet: Decrease of TS-induced NF-κB binding and promoter activity by ERK 1/2 inhibitors. A and B : extracted nuclear proteins without or with TS exposure were added into 96-well plates that had prebound NF-κB duplex DNA. Microwells bearing the biotinylated NF-κB DNA probe (1 pmol) were incubated with an increasing excess of the nonbiotinylated probe, containing either the wild-type (wt) or the mutated NF-κB-binding consensus sequence. Specific DNA binding activity for either p65 ( A ) or p50 ( B ) was determined by measured activity after incubation with respective primary antibody against p65 or p50. C : A549 cells were transiently transfected with a NF-κB-containing plasmid linked to the pGL3 basic vector and then treated without or with ERK 1/2 inhibitors. After TS exposure, cell supernatants were collected and assayed for luciferase activity as described in MATERIALS AND METHODS. Triplicate samples were harvested from each condition, and results are expressed as relative fold increase in luciferase activity compared with empty vector control. * P

    Techniques Used: Binding Assay, Activity Assay, Incubation, Sequencing, Transfection, Plasmid Preparation, Luciferase

    DNA binding activity of several TS-regulated TFs are susceptible to ERK 1/2 inhibition. A549 cells in a T25 flask were treated with TS as previously described and pretreated without inhibitor ( A ), with 10 μM U0126 ( B ), and with 20 μM PD98059 ( C ) for 2 h. The comparable signal intensity and contrast of the membranes were adjusted according to the alignment spots along the right and bottom edges, respectively. The TS-regulated TFs putatively mediated by ERK 1/2 were outlined with black boxes as indicated in  D . These results are representative of 3 independent experiments.
    Figure Legend Snippet: DNA binding activity of several TS-regulated TFs are susceptible to ERK 1/2 inhibition. A549 cells in a T25 flask were treated with TS as previously described and pretreated without inhibitor ( A ), with 10 μM U0126 ( B ), and with 20 μM PD98059 ( C ) for 2 h. The comparable signal intensity and contrast of the membranes were adjusted according to the alignment spots along the right and bottom edges, respectively. The TS-regulated TFs putatively mediated by ERK 1/2 were outlined with black boxes as indicated in D . These results are representative of 3 independent experiments.

    Techniques Used: Binding Assay, Activity Assay, Inhibition

    Effects of ERK 1/2 inhibitors on TS-regulated DNA binding activity in A549 cells. Cells were treated with and without TS and nuclear proteins were extracted and incubated with biotin-labeled probes corresponding to different TF binding sequences for EMSA as described in MATERIALS AND METHODS.  A : VDR.  B : RSRFC4.  C : ISCSP.  D : ISRE.  Lane 1  of each panel indicates free probes. DNA binding activity is represented without ( lane 2 ) and with ( lane 3 ) TS exposure in each panel.  Lane 4  of each panel represents binding activity after TS exposure but with 20× excess of cold probes added to the incubation.  Lane 5  (U0126) and  lane 6  (PD98059) are TS-exposed binding in the presence of ERK 1/2 inhibitors. These results are representative of 3 independent experiments. F p , free probe.
    Figure Legend Snippet: Effects of ERK 1/2 inhibitors on TS-regulated DNA binding activity in A549 cells. Cells were treated with and without TS and nuclear proteins were extracted and incubated with biotin-labeled probes corresponding to different TF binding sequences for EMSA as described in MATERIALS AND METHODS. A : VDR. B : RSRFC4. C : ISCSP. D : ISRE. Lane 1 of each panel indicates free probes. DNA binding activity is represented without ( lane 2 ) and with ( lane 3 ) TS exposure in each panel. Lane 4 of each panel represents binding activity after TS exposure but with 20× excess of cold probes added to the incubation. Lane 5 (U0126) and lane 6 (PD98059) are TS-exposed binding in the presence of ERK 1/2 inhibitors. These results are representative of 3 independent experiments. F p , free probe.

    Techniques Used: Binding Assay, Activity Assay, Incubation, Labeling

    TS-induced nuclear translocation of NF-κB and phosphorylation of IκB are mediated through ERK 1/2 signaling pathways. Overnight serum-starved A549 cells in a T25 flask were either directly exposed to 5 ml of freshly generated smoke for different time points as indicated ( A ) or pretreated with 10 μM U0126 and 20 μM PD98059 for 2 h followed by exposure to 5 ml of freshly generated smoke for 10 min ( B ). The membranes were probed with antibodies specific for phosphorylated IκB (pIκB), p65, p50, and GAPDH and developed with a chemiluminescence imaging system as described in MATERIALS AND METHODS. GAPDH intensity was used as internal control to normalize the sample loading. These results are representative of 3 independent experiments.
    Figure Legend Snippet: TS-induced nuclear translocation of NF-κB and phosphorylation of IκB are mediated through ERK 1/2 signaling pathways. Overnight serum-starved A549 cells in a T25 flask were either directly exposed to 5 ml of freshly generated smoke for different time points as indicated ( A ) or pretreated with 10 μM U0126 and 20 μM PD98059 for 2 h followed by exposure to 5 ml of freshly generated smoke for 10 min ( B ). The membranes were probed with antibodies specific for phosphorylated IκB (pIκB), p65, p50, and GAPDH and developed with a chemiluminescence imaging system as described in MATERIALS AND METHODS. GAPDH intensity was used as internal control to normalize the sample loading. These results are representative of 3 independent experiments.

    Techniques Used: Translocation Assay, Generated, Imaging

    22) Product Images from "Mechanical Stretch Increases the Proliferation While Inhibiting the Osteogenic Differentiation in Dental Pulp Stem Cells"

    Article Title: Mechanical Stretch Increases the Proliferation While Inhibiting the Osteogenic Differentiation in Dental Pulp Stem Cells

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2012.0099

    Phosphorylation in response to uniaxial stretch for the indicated periods in DPSCs.  (A)  Stretch-stimulated phosphorylation of Akt ( n =4).  (B)  Stretch-stimulated phosphorylation of p38 MAPK ( n =4).  (C)  Stretch-stimulated phosphorylation of ERK 1/2. Results
    Figure Legend Snippet: Phosphorylation in response to uniaxial stretch for the indicated periods in DPSCs. (A) Stretch-stimulated phosphorylation of Akt ( n =4). (B) Stretch-stimulated phosphorylation of p38 MAPK ( n =4). (C) Stretch-stimulated phosphorylation of ERK 1/2. Results

    Techniques Used:

    23) Product Images from "Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases"

    Article Title: Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases

    Journal: Nature Communications

    doi: 10.1038/ncomms10877

    Aldosterone-mediated cross-talk between the MR and the AT 1 R. ( a ) Representative immunoblots (upper panels) and densitometric quantitative analysis (lower panel) of multiple ( n =3) independent experiments to evaluate ERK 1/2 phosphorylation (pERK) as a ratio of activated ERK to total ERK (tERK) in neonatal rat ventricular myocytes (NRVMs) either unstimulated (Ns) or stimulated with aldosterone (Aldo 1 μM) for 15 min. Before Aldo treatment, myocytes were pre-treated with spironolactone (Spiro 10 μM) or losartan (Los 10 μM) for 30 min; * P
    Figure Legend Snippet: Aldosterone-mediated cross-talk between the MR and the AT 1 R. ( a ) Representative immunoblots (upper panels) and densitometric quantitative analysis (lower panel) of multiple ( n =3) independent experiments to evaluate ERK 1/2 phosphorylation (pERK) as a ratio of activated ERK to total ERK (tERK) in neonatal rat ventricular myocytes (NRVMs) either unstimulated (Ns) or stimulated with aldosterone (Aldo 1 μM) for 15 min. Before Aldo treatment, myocytes were pre-treated with spironolactone (Spiro 10 μM) or losartan (Los 10 μM) for 30 min; * P

    Techniques Used: Western Blot

    24) Product Images from "EP3 Prostanoid Receptor Isoforms Utilize Distinct Mechanisms to Regulate ERK 1/2 Activation"

    Article Title: EP3 Prostanoid Receptor Isoforms Utilize Distinct Mechanisms to Regulate ERK 1/2 Activation

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbalip.2009.01.021

    Phosphorylation of ERK 1/2 in HEK cells stably expressing either the EP 3-Ia , EP 3-II  or EP 3-III  receptor isoforms following treatment with increasing concentrations of PGE 2
    Figure Legend Snippet: Phosphorylation of ERK 1/2 in HEK cells stably expressing either the EP 3-Ia , EP 3-II or EP 3-III receptor isoforms following treatment with increasing concentrations of PGE 2

    Techniques Used: Stable Transfection, Expressing, IA

    Effects of various signaling pathway inhibitors on the PGE 2  induced phosphorylation of ERK 1/2 in HEK cells expressing either the EP 3-II  or EP 3-III  isoforms
    Figure Legend Snippet: Effects of various signaling pathway inhibitors on the PGE 2 induced phosphorylation of ERK 1/2 in HEK cells expressing either the EP 3-II or EP 3-III isoforms

    Techniques Used: Expressing

    25) Product Images from "ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury"

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    Journal: Neurochemical Research

    doi: 10.1007/s11064-018-2558-z

    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition. a and b Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Techniques Used: Activation Assay, Activity Assay, Inhibition, Western Blot, Cell Culture

    The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery
    Figure Legend Snippet: The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Techniques Used: Activity Assay, Functional Assay

    BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals.  a  Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126.  b  Quantification analysis of the infarct volume [n = 6, *P 
    Figure Legend Snippet: BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals. a Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126. b Quantification analysis of the infarct volume [n = 6, *P 

    Techniques Used: Inhibition, Activation Assay, Staining

    BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation.  a  Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 
    Figure Legend Snippet: BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation. a Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Techniques Used: Functional Assay, Inhibition, Activation Assay, Injection

    After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased.  a  and  b  Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm.  c  and  d  Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 
    Figure Legend Snippet: After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased. a and b Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm. c and d Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Techniques Used: Double Immunofluorescence Staining, Western Blot, Expressing

    BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury.  a  A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure.  b  and  c  Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 
    Figure Legend Snippet: BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury. a A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure. b and c Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Techniques Used: Western Blot, Expressing

    BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level.  a  Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level. a Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Techniques Used: Activity Assay, Western Blot, Expressing

    26) Product Images from "Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis"

    Article Title: Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191696

    iAβ expression leads to diminished EGF-dependent ERK1/2 phosphorylation. (A)  Immunoblot of phospho-ERK 1/2 and total-ERK 1/2 in VC or iAβ transduced HEK293 cells following EGF stimulation. While total-ERK 1/2 remains unaltered, decreased EGF dependent phospho-ERK 1/2 levels observed in iAβ-transduced cells relative to VC. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control.  (B)  Plot of the relative densitometry analysis in biological replicates depicting a relative decrease in phospho-ERK 1/2 levels with data shown as mean ± SE (n = 3) with statistical significance (**p
    Figure Legend Snippet: iAβ expression leads to diminished EGF-dependent ERK1/2 phosphorylation. (A) Immunoblot of phospho-ERK 1/2 and total-ERK 1/2 in VC or iAβ transduced HEK293 cells following EGF stimulation. While total-ERK 1/2 remains unaltered, decreased EGF dependent phospho-ERK 1/2 levels observed in iAβ-transduced cells relative to VC. Two-dimensional gel transformation of phospho-ERK 1/2 intensities performed and shown as histogram. Beta-actin used as loading control. (B) Plot of the relative densitometry analysis in biological replicates depicting a relative decrease in phospho-ERK 1/2 levels with data shown as mean ± SE (n = 3) with statistical significance (**p

    Techniques Used: Expressing, Two-Dimensional Gel Electrophoresis, Transformation Assay

    27) Product Images from "Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling"

    Article Title: Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200317295

    Generation of cardiac-specific transgenic mice expressing dominant-negative mutants of p38α, MKK3, and MKK6. ( a ) Western blot analysis with Ab’s against p38α, p38β, MKK3, and MKK6 from nontransgenic (NTG) and transgenic (TG) hearts regulated by the α-MHC promoter ( b ) Western blot analysis of p38 phosphorylation in the hearts of nontransgenic (wild-type littermates) or dnMKK3 and dnMKK6 transgenic mice injected for 30 min with either PBS or PE (10 mg/kg). To verify specificity, phospho–ERK-1/2 (phos-ERK-1/2) and phospho-JNK (phos-JNK) were also assayed. The asterisks show the reduced phosphorylation of p38 at baseline (PBS) and in response to PE stimulation. ( c ) p38 immune kinase assay from PBS- and PE-injected nontransgenic mice or dnp38α, dnMKK3, and dnMKK6 mice. Thirty minutes after stimulation, the hearts were removed and phosphorylation of MBP was monitored by immune kinase assay with p38-specific Ab. Three independent p38 immune kinase assays showed increased activity in NTG hearts and dnMKK6 hearts. Veh, vehicle. ( d ) Western blot analysis of MAPKAPK2 phosphorylation (phos-MKAPK2), a direct p38 target, in the hearts of nontransgenic (wild-type littermates) or each of the dominant-negative transgenic mice after PE stimulation (10 mg/kg). All three dominant-negative strategies significantly reduced p38 kinase activity in c and d ( # P
    Figure Legend Snippet: Generation of cardiac-specific transgenic mice expressing dominant-negative mutants of p38α, MKK3, and MKK6. ( a ) Western blot analysis with Ab’s against p38α, p38β, MKK3, and MKK6 from nontransgenic (NTG) and transgenic (TG) hearts regulated by the α-MHC promoter ( b ) Western blot analysis of p38 phosphorylation in the hearts of nontransgenic (wild-type littermates) or dnMKK3 and dnMKK6 transgenic mice injected for 30 min with either PBS or PE (10 mg/kg). To verify specificity, phospho–ERK-1/2 (phos-ERK-1/2) and phospho-JNK (phos-JNK) were also assayed. The asterisks show the reduced phosphorylation of p38 at baseline (PBS) and in response to PE stimulation. ( c ) p38 immune kinase assay from PBS- and PE-injected nontransgenic mice or dnp38α, dnMKK3, and dnMKK6 mice. Thirty minutes after stimulation, the hearts were removed and phosphorylation of MBP was monitored by immune kinase assay with p38-specific Ab. Three independent p38 immune kinase assays showed increased activity in NTG hearts and dnMKK6 hearts. Veh, vehicle. ( d ) Western blot analysis of MAPKAPK2 phosphorylation (phos-MKAPK2), a direct p38 target, in the hearts of nontransgenic (wild-type littermates) or each of the dominant-negative transgenic mice after PE stimulation (10 mg/kg). All three dominant-negative strategies significantly reduced p38 kinase activity in c and d ( # P

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Dominant Negative Mutation, Western Blot, Injection, Kinase Assay, Activity Assay

    28) Product Images from "Safranal Alleviates Dextran Sulfate Sodium-Induced Colitis and Suppresses Macrophage-Mediated Inflammation"

    Article Title: Safranal Alleviates Dextran Sulfate Sodium-Induced Colitis and Suppresses Macrophage-Mediated Inflammation

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2019.01281

    Safranal inhibits the NF-κB and MAPK pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cells were pretreated with Safranal for 1 h prior to LPS treatment. The levels of phosphorylated IKK, IκBα, ERK, JNK, and p38 were measured after treatment with 1 μg/ml LPS for 15 min. Signaling proteins in the (A) NF-κB pathway and (B) MAPK pathway were analyzed using Western blot and normalized to β-actin. The data shown are representative of three independent experiments and indicate the means ± SEM. ### p
    Figure Legend Snippet: Safranal inhibits the NF-κB and MAPK pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cells were pretreated with Safranal for 1 h prior to LPS treatment. The levels of phosphorylated IKK, IκBα, ERK, JNK, and p38 were measured after treatment with 1 μg/ml LPS for 15 min. Signaling proteins in the (A) NF-κB pathway and (B) MAPK pathway were analyzed using Western blot and normalized to β-actin. The data shown are representative of three independent experiments and indicate the means ± SEM. ### p

    Techniques Used: Western Blot

    Safranal reduced cytokine production via suppression of MAPK and NF-κB proteins in colonic tissues from colitis mice. Colons were homogenized for measurement of IL-6 and TNF-α levels (A , B) . The phosphorylation of JNK, ERK, p38, and IκBα was analyzed using Western blotting (C) and the density of proteins was calculated and normalized to β-actin. All data are the means ± SEM, n = 10. # p
    Figure Legend Snippet: Safranal reduced cytokine production via suppression of MAPK and NF-κB proteins in colonic tissues from colitis mice. Colons were homogenized for measurement of IL-6 and TNF-α levels (A , B) . The phosphorylation of JNK, ERK, p38, and IκBα was analyzed using Western blotting (C) and the density of proteins was calculated and normalized to β-actin. All data are the means ± SEM, n = 10. # p

    Techniques Used: Mouse Assay, Western Blot

    29) Product Images from "Loss of PTEN stabilizes the lipid modifying enzyme cytosolic phospholipase A2α via AKT in prostate cancer cells"

    Article Title: Loss of PTEN stabilizes the lipid modifying enzyme cytosolic phospholipase A2α via AKT in prostate cancer cells

    Journal: Oncotarget

    doi:

    Effect of pAKT on cPLA 2 α is independent of ERK 1/2 (a) LNCaP cells stably transfected with Dox-controlled inducible cPLA 2 α expression system (LNCaP- i -cPLA 2 α) were treated with Dox (100 ng/mL, 24 h), followed by incubation with MEK inhibitor U0126 (5 μM, 1 h). The cells were harvested 24 h later. (b) LNCaP- i- cPLA 2 α cells induced with Dox for 1 h and transfected with Myr-AKT or empty vector (2 μg, 24 h), followed by incubation with U0126 (5 μM, 1 h). The cells were harvested 24 h later. All immunoblotting results are typical of 3 independent experiments.
    Figure Legend Snippet: Effect of pAKT on cPLA 2 α is independent of ERK 1/2 (a) LNCaP cells stably transfected with Dox-controlled inducible cPLA 2 α expression system (LNCaP- i -cPLA 2 α) were treated with Dox (100 ng/mL, 24 h), followed by incubation with MEK inhibitor U0126 (5 μM, 1 h). The cells were harvested 24 h later. (b) LNCaP- i- cPLA 2 α cells induced with Dox for 1 h and transfected with Myr-AKT or empty vector (2 μg, 24 h), followed by incubation with U0126 (5 μM, 1 h). The cells were harvested 24 h later. All immunoblotting results are typical of 3 independent experiments.

    Techniques Used: Stable Transfection, Transfection, Expressing, Incubation, Plasmid Preparation

    30) Product Images from "Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor"

    Article Title: Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.612630

    MAPK signaling of FPR2 and N333-stop. A  and  B , HEK293 cells stably expressing either N-terminally FLAG-tagged WT FPR2 or N333-stop ( N333 ) were serum-starved 4 h prior to experimentation. Cells were untreated or stimulated with 500 n m  W peptide for 5, 10, and 30 min, lysed, separated by SDS-PAGE, and probed for phospho-ERK 1/2 ( A ) or JNK ( B ), and then stripped and reprobed for total ERK 1/2 and JNK.  C , for resensitization, phospho-ERK 1/2 was investigated where cells were either untreated, stimulated for 5 min, pretreated with agonist for 30 min, and then re-challenged with vehicle or drug (desensitized) or pretreated for 30 min, washed, and allowed to recover for 90 min at 37 °C before a final re-challenge for 5 min with agonist or vehicle (resensitization and recycling). Representative blots are shown of at least three independent experiments.
    Figure Legend Snippet: MAPK signaling of FPR2 and N333-stop. A and B , HEK293 cells stably expressing either N-terminally FLAG-tagged WT FPR2 or N333-stop ( N333 ) were serum-starved 4 h prior to experimentation. Cells were untreated or stimulated with 500 n m W peptide for 5, 10, and 30 min, lysed, separated by SDS-PAGE, and probed for phospho-ERK 1/2 ( A ) or JNK ( B ), and then stripped and reprobed for total ERK 1/2 and JNK. C , for resensitization, phospho-ERK 1/2 was investigated where cells were either untreated, stimulated for 5 min, pretreated with agonist for 30 min, and then re-challenged with vehicle or drug (desensitized) or pretreated for 30 min, washed, and allowed to recover for 90 min at 37 °C before a final re-challenge for 5 min with agonist or vehicle (resensitization and recycling). Representative blots are shown of at least three independent experiments.

    Techniques Used: Stable Transfection, Expressing, SDS Page

    31) Product Images from "Myocardial ischemic postconditioning against ischemia-reperfusion is impaired in ob/ob mice"

    Article Title: Myocardial ischemic postconditioning against ischemia-reperfusion is impaired in ob/ob mice

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00379.2008

    Western blot analysis of Akt and its phosphorylated form at Ser 473 (panel A), ERK 1/2 and its phosphorylated forms at Thr 202 and Tyr 204 (panel B), p70S6K1 and its phosphorylated form at Thr 389 due to the effect of Akt (panel C), p70S6K1 and its phosphorylated form at Thr 421 and Ser 424 due to the effects of ERK1/2 (panel D), AMPK and its phosphorylated form (panel E), PTEN (panel F), MKP-3 (panel G) and PP2C (panel H) in wild-type (WT) and ob/ob mice performed at baseline ( i.e ., in the absence of ischemia-reperfusion). Values are expressed as mean ± SEM (n=5 per group for WT mice and n=5 per group for ob/ob mice). * p
    Figure Legend Snippet: Western blot analysis of Akt and its phosphorylated form at Ser 473 (panel A), ERK 1/2 and its phosphorylated forms at Thr 202 and Tyr 204 (panel B), p70S6K1 and its phosphorylated form at Thr 389 due to the effect of Akt (panel C), p70S6K1 and its phosphorylated form at Thr 421 and Ser 424 due to the effects of ERK1/2 (panel D), AMPK and its phosphorylated form (panel E), PTEN (panel F), MKP-3 (panel G) and PP2C (panel H) in wild-type (WT) and ob/ob mice performed at baseline ( i.e ., in the absence of ischemia-reperfusion). Values are expressed as mean ± SEM (n=5 per group for WT mice and n=5 per group for ob/ob mice). * p

    Techniques Used: Western Blot, Mouse Assay

    Western blot analysis of p70S6K1 and its phosphorylated forms. Panel A: Thr 389 due to the effect of Akt; panel B: Thr 421 and Ser 424 due to the effects of ERK 1/2 in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). * p
    Figure Legend Snippet: Western blot analysis of p70S6K1 and its phosphorylated forms. Panel A: Thr 389 due to the effect of Akt; panel B: Thr 421 and Ser 424 due to the effects of ERK 1/2 in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). * p

    Techniques Used: Western Blot, Mouse Assay

    Western blot analysis of Akt and its phosphorylated form at Ser 473 (panel A) as well as ERK 1/2 and its phosphorylated forms at Thr 202 and Tyr 204 (panel B) in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). * p
    Figure Legend Snippet: Western blot analysis of Akt and its phosphorylated form at Ser 473 (panel A) as well as ERK 1/2 and its phosphorylated forms at Thr 202 and Tyr 204 (panel B) in wild-type (WT) and ob/ob mice submitted to 30 min of coronary artery occlusion and 10 min of reperfusion in the absence (Control) or presence of postconditioning (IPCD). Values are expressed as mean ± SEM (n=3 per group for WT mice and n=4 per group for ob/ob mice). * p

    Techniques Used: Western Blot, Mouse Assay

    32) Product Images from "ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury"

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    Journal: Neurochemical Research

    doi: 10.1007/s11064-018-2558-z

    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition. a and b Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Techniques Used: Activation Assay, Activity Assay, Inhibition, Western Blot, Cell Culture

    The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery
    Figure Legend Snippet: The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Techniques Used: Activity Assay, Functional Assay

    BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals.  a  Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126.  b  Quantification analysis of the infarct volume [n = 6, *P 
    Figure Legend Snippet: BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals. a Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126. b Quantification analysis of the infarct volume [n = 6, *P 

    Techniques Used: Inhibition, Activation Assay, Staining

    BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation.  a  Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 
    Figure Legend Snippet: BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation. a Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Techniques Used: Functional Assay, Inhibition, Activation Assay, Injection

    After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased.  a  and  b  Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm.  c  and  d  Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 
    Figure Legend Snippet: After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased. a and b Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm. c and d Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Techniques Used: Double Immunofluorescence Staining, Western Blot, Expressing

    BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury.  a  A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure.  b  and  c  Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 
    Figure Legend Snippet: BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury. a A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure. b and c Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Techniques Used: Western Blot, Expressing

    BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level.  a  Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 
    Figure Legend Snippet: BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level. a Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Techniques Used: Activity Assay, Western Blot, Expressing

    33) Product Images from "Inhibitory Activity of Bevacizumab to Differentiation of Retinoblastoma Cells"

    Article Title: Inhibitory Activity of Bevacizumab to Differentiation of Retinoblastoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033456

    Bevacizumab-induced Inhibition of Differentiation of Retinoblastoma Cells through Blockade of ERK 1/2 Activation. SNUOT-Rb1 cells were treated with 0.1% BSA or 1 mg/ml bevacizumab. ERK 1/2, phospho-ERK 1/2, Akt, and phospho-Akt were detected by Western blot analysis. β-actin was served as a loading control. Each figure is representative ones from three independent experiments. Quantitative analysis was performed by measuring protein expression relative to the controls. Each value represents means ± SE from three independent experiments (*P
    Figure Legend Snippet: Bevacizumab-induced Inhibition of Differentiation of Retinoblastoma Cells through Blockade of ERK 1/2 Activation. SNUOT-Rb1 cells were treated with 0.1% BSA or 1 mg/ml bevacizumab. ERK 1/2, phospho-ERK 1/2, Akt, and phospho-Akt were detected by Western blot analysis. β-actin was served as a loading control. Each figure is representative ones from three independent experiments. Quantitative analysis was performed by measuring protein expression relative to the controls. Each value represents means ± SE from three independent experiments (*P

    Techniques Used: Inhibition, Activation Assay, Western Blot, Expressing

    34) Product Images from "Ghrelin's Effects on Proinflammatory Cytokine Mediated Apoptosis and Their Impact on β-Cell Functionality"

    Article Title: Ghrelin's Effects on Proinflammatory Cytokine Mediated Apoptosis and Their Impact on β-Cell Functionality

    Journal: International Journal of Endocrinology

    doi: 10.1155/2015/235727

    Western blot for phosphorylated AKT (a, c, and e) and ERK 1/2 (b, d, and f). Immunoblotting was performed to quantify phosphorylation level or AKT an ERK 1/2 treated with proinflammatory cytokines during 15 (a and d), 30 (b and e), and 60 min (c and f) alone or in presence of ghrelin. Panels below the graph show representative images of immunoblots. Results are expressed as mean ± SEM of phosphorylated to total protein ratio and referred to control cultures (c). Values were obtained from 4 independent experiments.  * p
    Figure Legend Snippet: Western blot for phosphorylated AKT (a, c, and e) and ERK 1/2 (b, d, and f). Immunoblotting was performed to quantify phosphorylation level or AKT an ERK 1/2 treated with proinflammatory cytokines during 15 (a and d), 30 (b and e), and 60 min (c and f) alone or in presence of ghrelin. Panels below the graph show representative images of immunoblots. Results are expressed as mean ± SEM of phosphorylated to total protein ratio and referred to control cultures (c). Values were obtained from 4 independent experiments. * p

    Techniques Used: Western Blot

    35) Product Images from "Endothelial Progenitor Cells and a Stromal Cell–derived Factor-1α Analogue Synergistically Improve Survival in Sepsis"

    Article Title: Endothelial Progenitor Cells and a Stromal Cell–derived Factor-1α Analogue Synergistically Improve Survival in Sepsis

    Journal: American Journal of Respiratory and Critical Care Medicine

    doi: 10.1164/rccm.201312-2163OC

    Effects of CTCE on signaling protein expression in endothelial progenitor cells (EPCs). EPCs were treated with CTCE (1 μg/ml) for different time intervals. ( A ) Phospho-ERK 1/2, ( B ) Phospho-AKT, ( C ) Phospho-GSK3β, ( D ) Phospho-eNOS, and ( E ) Spred-1 were determined by Western blot. ( F  and  G ) EPCs were transfected with control or miR-126 inhibitor and treated with CTCE (1 μg/ml). ERK 1/2 activation and EPC proliferation were determined. The statistical analysis was performed on densitometry scans. * P
    Figure Legend Snippet: Effects of CTCE on signaling protein expression in endothelial progenitor cells (EPCs). EPCs were treated with CTCE (1 μg/ml) for different time intervals. ( A ) Phospho-ERK 1/2, ( B ) Phospho-AKT, ( C ) Phospho-GSK3β, ( D ) Phospho-eNOS, and ( E ) Spred-1 were determined by Western blot. ( F and G ) EPCs were transfected with control or miR-126 inhibitor and treated with CTCE (1 μg/ml). ERK 1/2 activation and EPC proliferation were determined. The statistical analysis was performed on densitometry scans. * P

    Techniques Used: Expressing, Western Blot, Transfection, Activation Assay

    36) Product Images from "Refined Deep-Sea Water Suppresses Inflammatory Responses via the MAPK/AP-1 and NF-κB Signaling Pathway in LPS-Treated RAW 264.7 Macrophage Cells"

    Article Title: Refined Deep-Sea Water Suppresses Inflammatory Responses via the MAPK/AP-1 and NF-κB Signaling Pathway in LPS-Treated RAW 264.7 Macrophage Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18112282

    Effect of RDSW on phosphorylation of IκB, nuclear translocation of NF-κB, and activations of ERK 1/2 and JNK 1/2 in LPS-induced RAW 264.7 macrophage cells. The cells were pre-treated with RDSW for 24 h prior to the stimulation of LPS (1 µg/mL) for various incubation times (IκB; 15 min, NF-κB, ERK 1/2 and JNK 1/2; 30 min). RDSW inhibited LPS-induced ( A ) phosphorylation of IκB, ( B ) nuclear translocation of NF-κB, and activations of ( C ) ERK 1/2 and ( D ) JNK 1/2. β-actin was used as the internal control for the whole cell lysate and cytosolic fraction, and Lamin B1 was used as the internal control for the nuclear fraction. Band densities of p-IκB, NF-κB, p-ERK 1/2, and p-JNK 1/2 were normalized with IκB, Lamin B1, ERK 1/2 and JNK 1/2, respectively. All experiments were performed in triplicate, and all data are shown as the mean ± SD. **  p
    Figure Legend Snippet: Effect of RDSW on phosphorylation of IκB, nuclear translocation of NF-κB, and activations of ERK 1/2 and JNK 1/2 in LPS-induced RAW 264.7 macrophage cells. The cells were pre-treated with RDSW for 24 h prior to the stimulation of LPS (1 µg/mL) for various incubation times (IκB; 15 min, NF-κB, ERK 1/2 and JNK 1/2; 30 min). RDSW inhibited LPS-induced ( A ) phosphorylation of IκB, ( B ) nuclear translocation of NF-κB, and activations of ( C ) ERK 1/2 and ( D ) JNK 1/2. β-actin was used as the internal control for the whole cell lysate and cytosolic fraction, and Lamin B1 was used as the internal control for the nuclear fraction. Band densities of p-IκB, NF-κB, p-ERK 1/2, and p-JNK 1/2 were normalized with IκB, Lamin B1, ERK 1/2 and JNK 1/2, respectively. All experiments were performed in triplicate, and all data are shown as the mean ± SD. ** p

    Techniques Used: Translocation Assay, Incubation

    Inhibitory effects of RDSW on LPS-induced inflammatory responses were mediated through the inhibition of the ERK 1/2 and JNK 1/2 signaling pathways. The inhibitory effects of RDSW on LPS-induced ( A ) c-Jun and c-Fos expressions. The involvements of ERK 1/2 and JNK 1/2 signaling pathways in LPS-induced inflammatory response were confirmed by blocking each pathway with a specific inhibitor. The effects of ( B ) 10 μM U0126 (a MEK 1/2 inhibitor) or ( C ) 10 μM SP600125 (a JNK inhibitor) on the expressions of c-Jun and c-Fos in LPS-treated RAW 264.7 macrophage cells. The cells were pre-treated with RDSW for 22 h, and then treated with 10 μM U0126 (a MEK 1/2 inhibitor) or 10 μM SP600125 (a JNK inhibitor) for 2 h prior to LPS stimulation for 1 h. Band density of each sample was normalized with β-actin. All experiments were performed in triplicates, and all data are shown as the mean ± SD. **  p
    Figure Legend Snippet: Inhibitory effects of RDSW on LPS-induced inflammatory responses were mediated through the inhibition of the ERK 1/2 and JNK 1/2 signaling pathways. The inhibitory effects of RDSW on LPS-induced ( A ) c-Jun and c-Fos expressions. The involvements of ERK 1/2 and JNK 1/2 signaling pathways in LPS-induced inflammatory response were confirmed by blocking each pathway with a specific inhibitor. The effects of ( B ) 10 μM U0126 (a MEK 1/2 inhibitor) or ( C ) 10 μM SP600125 (a JNK inhibitor) on the expressions of c-Jun and c-Fos in LPS-treated RAW 264.7 macrophage cells. The cells were pre-treated with RDSW for 22 h, and then treated with 10 μM U0126 (a MEK 1/2 inhibitor) or 10 μM SP600125 (a JNK inhibitor) for 2 h prior to LPS stimulation for 1 h. Band density of each sample was normalized with β-actin. All experiments were performed in triplicates, and all data are shown as the mean ± SD. ** p

    Techniques Used: Inhibition, Blocking Assay

    37) Product Images from "Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3"

    Article Title: Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0140583

    Transactivation of EGF receptors in LPA-induced ERK 1/2 phosphorylation. Cells overexpressing LPA 1  (panels A and D), LPA 2  (panels B and E) or LPA 3  (panels C and F) receptors were incubated in the absence or presence of 10 μM AG1478 (AG) for 30 min and then challenged with 1 μM LPA (panels A-C) or 100 ng/ml EGF (panels D-F) for 5 min; incubation was terminated and phospho-ERK 1/2 (pERK) and total ERK 1/2 (ERK) were assayed by Western blotting. Plotted are the increases in phospho-ERK 1/2 as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative Western blots are presented for the different receptor subtypes. *p
    Figure Legend Snippet: Transactivation of EGF receptors in LPA-induced ERK 1/2 phosphorylation. Cells overexpressing LPA 1 (panels A and D), LPA 2 (panels B and E) or LPA 3 (panels C and F) receptors were incubated in the absence or presence of 10 μM AG1478 (AG) for 30 min and then challenged with 1 μM LPA (panels A-C) or 100 ng/ml EGF (panels D-F) for 5 min; incubation was terminated and phospho-ERK 1/2 (pERK) and total ERK 1/2 (ERK) were assayed by Western blotting. Plotted are the increases in phospho-ERK 1/2 as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative Western blots are presented for the different receptor subtypes. *p

    Techniques Used: Incubation, Western Blot

    Effect of LPA on ERK 1/2 phosphorylation. Cells overexpressing LPA 1  (black, circles), LPA 2  (blue, squares) or LPA 3  (red, triangles) receptors were incubated for the times indicated in the presence of 1 μM LPA, incubation was terminated and phospho-ERK 1/2 (pERK) and total ERK 1/2 (ERK) were assayed by Western blotting. Plotted are the increases in phospho-ERK 1/2 as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative Western blots are presented for the different receptor subtypes.
    Figure Legend Snippet: Effect of LPA on ERK 1/2 phosphorylation. Cells overexpressing LPA 1 (black, circles), LPA 2 (blue, squares) or LPA 3 (red, triangles) receptors were incubated for the times indicated in the presence of 1 μM LPA, incubation was terminated and phospho-ERK 1/2 (pERK) and total ERK 1/2 (ERK) were assayed by Western blotting. Plotted are the increases in phospho-ERK 1/2 as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative Western blots are presented for the different receptor subtypes.

    Techniques Used: Incubation, Western Blot

    38) Product Images from "Protein disulfide isomerase expression increases in resistance arteries during hypertension development. Effects on Nox1 NADPH oxidase signaling"

    Article Title: Protein disulfide isomerase expression increases in resistance arteries during hypertension development. Effects on Nox1 NADPH oxidase signaling

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2015.00024

    PDI inhibition decreases Ang II induced ERK 1/2 activation in mesenteric resistance arteries of Wistar and SHR . Ang II rapidly induced ERK 1/2 phosphorylation (p-ERK; 5 min) and this activation is increased in SHR mesenteric arteries as compared to Wistar. PDI inhibition (Bacitracin 0.5 mmol/L) significantly abolished p-ERK 1/2 in mesenteric arteries of both strains. Upper panel shows a representative blot for three experiments. Bar graph indicates relative quantification of p-ERK content expressed as the ratio between pERK and ERK 1/2 and normalized to the vehicle group, taken as 100%. Results are expressed as percentage of Wistar vehicle and represent mean ± SEM.  * p
    Figure Legend Snippet: PDI inhibition decreases Ang II induced ERK 1/2 activation in mesenteric resistance arteries of Wistar and SHR . Ang II rapidly induced ERK 1/2 phosphorylation (p-ERK; 5 min) and this activation is increased in SHR mesenteric arteries as compared to Wistar. PDI inhibition (Bacitracin 0.5 mmol/L) significantly abolished p-ERK 1/2 in mesenteric arteries of both strains. Upper panel shows a representative blot for three experiments. Bar graph indicates relative quantification of p-ERK content expressed as the ratio between pERK and ERK 1/2 and normalized to the vehicle group, taken as 100%. Results are expressed as percentage of Wistar vehicle and represent mean ± SEM. * p

    Techniques Used: Inhibition, Activation Assay

    39) Product Images from "Evidence for functional selectivity in TUDC- and norUDCA-induced signal transduction via α5β1 integrin towards choleresis"

    Article Title: Evidence for functional selectivity in TUDC- and norUDCA-induced signal transduction via α5β1 integrin towards choleresis

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-62326-y

    Model of α 5 β 1 integrin activation-dependent differential bile acid signaling. Activation of α 5 β 1 integrin with the less efficacious nor UDCA results in the formation of FAK Y397-P , which leads to c-Src- and PI3-K-dependent Erk-1/2 activation. When α 5 β 1 integrin is activated by the more efficacious TUDC, higher levels of FAK Y397-P result, which, in addition, trigger a slower activation of Erk-1/-2 via PI3-K in a c-Src-independent manner 7 .
    Figure Legend Snippet: Model of α 5 β 1 integrin activation-dependent differential bile acid signaling. Activation of α 5 β 1 integrin with the less efficacious nor UDCA results in the formation of FAK Y397-P , which leads to c-Src- and PI3-K-dependent Erk-1/2 activation. When α 5 β 1 integrin is activated by the more efficacious TUDC, higher levels of FAK Y397-P result, which, in addition, trigger a slower activation of Erk-1/-2 via PI3-K in a c-Src-independent manner 7 .

    Techniques Used: Activation Assay

    TUDC-induced dual activation of Erk-1/-2 and p38 MAPK and Bsep insertion into the canalicular membrane are dependent on EGFR phosphorylation. Rat livers were perfused with TUDC (20 µmol/l) for up to 60 min. When indicated, AG1478 (1 µmol/l) was added 30 min prior to TUDC to the perfusate. ( a ) Phosphorylation of Erk-1/-2 and p38 MAPK was analyzed by use of specific antibodies. Total Erk-1/-2 or p38 MAPK , respectively, served as loading controls. ( b ) Western blots were analyzed densitometrically. Phosphorylation level at t = 0 min was set to 1. Representative blots and statistics (mean ± SEM) of at least three independent perfusion experiments are shown. TUDC induced a significant increase in Erk-1/-2 and p38 MAPK phosphorylation (* p
    Figure Legend Snippet: TUDC-induced dual activation of Erk-1/-2 and p38 MAPK and Bsep insertion into the canalicular membrane are dependent on EGFR phosphorylation. Rat livers were perfused with TUDC (20 µmol/l) for up to 60 min. When indicated, AG1478 (1 µmol/l) was added 30 min prior to TUDC to the perfusate. ( a ) Phosphorylation of Erk-1/-2 and p38 MAPK was analyzed by use of specific antibodies. Total Erk-1/-2 or p38 MAPK , respectively, served as loading controls. ( b ) Western blots were analyzed densitometrically. Phosphorylation level at t = 0 min was set to 1. Representative blots and statistics (mean ± SEM) of at least three independent perfusion experiments are shown. TUDC induced a significant increase in Erk-1/-2 and p38 MAPK phosphorylation (* p

    Techniques Used: Activation Assay, Western Blot

    nor UDCA-induced activation of Erk-1/-2, p38 MAPK and Src. Rat livers were perfused with nor UDCA (20 µmol/l) for up to 60 min. Liver samples were taken at the time points indicated. The integrin antagonistic peptide (G RGD SP, 10 µmol/l), the inactive control peptide (G RAD SP, 10 µmol/l), the PI3-K inhibitor wortmannin (100 nmol/l), and the Src inhibitor PP-2 (250 nmol/l) were added 30 min prior to the addition of nor UDCA. Activation of Erk-1/-2, p38 MAPK and c-Src was analyzed by ( a,b ) Western blot using specific antibodies and ( c,d ) subsequent densitometric analysis. Total Erk-1/-2, total p38 MAPK , and total c-Src served as respective loading control. Phosphorylation at t = 0 min was arbitrarily set as 1. Densitometric analyses (means ± SEM) and representative blots of at least three independent perfusion experiments are shown. * p
    Figure Legend Snippet: nor UDCA-induced activation of Erk-1/-2, p38 MAPK and Src. Rat livers were perfused with nor UDCA (20 µmol/l) for up to 60 min. Liver samples were taken at the time points indicated. The integrin antagonistic peptide (G RGD SP, 10 µmol/l), the inactive control peptide (G RAD SP, 10 µmol/l), the PI3-K inhibitor wortmannin (100 nmol/l), and the Src inhibitor PP-2 (250 nmol/l) were added 30 min prior to the addition of nor UDCA. Activation of Erk-1/-2, p38 MAPK and c-Src was analyzed by ( a,b ) Western blot using specific antibodies and ( c,d ) subsequent densitometric analysis. Total Erk-1/-2, total p38 MAPK , and total c-Src served as respective loading control. Phosphorylation at t = 0 min was arbitrarily set as 1. Densitometric analyses (means ± SEM) and representative blots of at least three independent perfusion experiments are shown. * p

    Techniques Used: Activation Assay, Western Blot

    Comparison between nor UDCA- and TUDC-induced Erk-1/-2, p38 MAPK and EGFR activation. Rat livers were perfused with nor UDCA or TUDC (20 µmol/l each) for up to 60 min as described in “Experimental Procedures”. Liver samples were taken at the time points indicated. Phosphorylation of Erk-1/-2, p38 MAPK , and EGFR tyrosine residues Tyr 845 , Tyr 1045 , and Tyr 1173 was analyzed by ( a ) Western blot using specific antibodies and ( b ) subsequent densitometric analysis (black squares, nor UDCA; gray squares, TUDC). Total Erk-1/-2, total p38 MAPK , and total EGFR served as respective loading controls. Phosphorylation at t = 0 was arbitrarily set to 1. Data represent the mean (mean ± SEM) of at least three independent experiments; * p
    Figure Legend Snippet: Comparison between nor UDCA- and TUDC-induced Erk-1/-2, p38 MAPK and EGFR activation. Rat livers were perfused with nor UDCA or TUDC (20 µmol/l each) for up to 60 min as described in “Experimental Procedures”. Liver samples were taken at the time points indicated. Phosphorylation of Erk-1/-2, p38 MAPK , and EGFR tyrosine residues Tyr 845 , Tyr 1045 , and Tyr 1173 was analyzed by ( a ) Western blot using specific antibodies and ( b ) subsequent densitometric analysis (black squares, nor UDCA; gray squares, TUDC). Total Erk-1/-2, total p38 MAPK , and total EGFR served as respective loading controls. Phosphorylation at t = 0 was arbitrarily set to 1. Data represent the mean (mean ± SEM) of at least three independent experiments; * p

    Techniques Used: Activation Assay, Western Blot

    40) Product Images from "Phasic Phosphorylation of Caldesmon and ERK 1/2 during Contractions in Human Myometrium"

    Article Title: Phasic Phosphorylation of Caldesmon and ERK 1/2 during Contractions in Human Myometrium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021542

    h-CaD and ERK 1/2 expression in NIL and L term human myometrium. NIL and L myometrial protein extracts were separated by SDS-PAGE and western transferred onto nitrocellulose membrane. Membranes were probed with antibodies against phospho-h-CaD (1∶2000), total h-CaD (1∶2000), phospho-ERK 1/2 (1∶2000), total ERK 1/2 (1∶2000) and α-SMA (1∶10000). A total 8×NIL and 8×L samples were analysed. Representative image demonstrates detection of these proteins on a single blot containing 4×NIL and 4×L myometrial samples, as well as a calibrator sample that was included to allow densitometric comparison across separate blots. M r  = relative molecular mass×1000.
    Figure Legend Snippet: h-CaD and ERK 1/2 expression in NIL and L term human myometrium. NIL and L myometrial protein extracts were separated by SDS-PAGE and western transferred onto nitrocellulose membrane. Membranes were probed with antibodies against phospho-h-CaD (1∶2000), total h-CaD (1∶2000), phospho-ERK 1/2 (1∶2000), total ERK 1/2 (1∶2000) and α-SMA (1∶10000). A total 8×NIL and 8×L samples were analysed. Representative image demonstrates detection of these proteins on a single blot containing 4×NIL and 4×L myometrial samples, as well as a calibrator sample that was included to allow densitometric comparison across separate blots. M r  = relative molecular mass×1000.

    Techniques Used: Expressing, SDS Page, Western Blot

    Densitometric analysis of h-CaD and ERK2 expression in NIL and L myometrium. Densitometric analysis was performed for 8×NIL and 8×L myometrial samples following immunodetection of phospho-h-CaD, total h-CaD, phospho-ERK 1/2, total ERK 1/2 and α-SMA. (A) Onset of labor was associated with a statistically significant, 2-fold up-regulation in h-CaD phosphorylation, whilst (B) total h-CaD expression remained unchanged. (C) ERK2 phosphorylation remained unchanged following the onset of labor, however (D) total ERK2 expression underwent a statistically significant 1.4-fold up-regulation in association with the onset of labor. *p
    Figure Legend Snippet: Densitometric analysis of h-CaD and ERK2 expression in NIL and L myometrium. Densitometric analysis was performed for 8×NIL and 8×L myometrial samples following immunodetection of phospho-h-CaD, total h-CaD, phospho-ERK 1/2, total ERK 1/2 and α-SMA. (A) Onset of labor was associated with a statistically significant, 2-fold up-regulation in h-CaD phosphorylation, whilst (B) total h-CaD expression remained unchanged. (C) ERK2 phosphorylation remained unchanged following the onset of labor, however (D) total ERK2 expression underwent a statistically significant 1.4-fold up-regulation in association with the onset of labor. *p

    Techniques Used: Expressing, Immunodetection

    h-CaD and ERK 1/2 phosphorylation during myometrial contractions  in vitro . (A) Myometrial strips were snap frozen at specific stages during the development of contractions. The tissue was then pulverised and subjected to protein extraction. Protein (20 µg) was separated by 1D PAGE and transferred to a nitrocellulose membrane. (B) Membranes were probed with antibodies against phospho-h-CaD (1∶2000), total h-CaD (1∶2000), phospho-ERK 1/2 (1∶2000) and total ERK 1/2 (1∶2000), as well as α-SMA (1∶10000), representative images shown of 5 replicates. (C) Non-parametric analyses of the non-normalised raw data revealed a statistically significant 2-fold increase in h-CaD phosphorylation during the transition from point 1 to point 2. No significant difference was observed between contraction points 1 and 3 or 2 and 3. *p
    Figure Legend Snippet: h-CaD and ERK 1/2 phosphorylation during myometrial contractions in vitro . (A) Myometrial strips were snap frozen at specific stages during the development of contractions. The tissue was then pulverised and subjected to protein extraction. Protein (20 µg) was separated by 1D PAGE and transferred to a nitrocellulose membrane. (B) Membranes were probed with antibodies against phospho-h-CaD (1∶2000), total h-CaD (1∶2000), phospho-ERK 1/2 (1∶2000) and total ERK 1/2 (1∶2000), as well as α-SMA (1∶10000), representative images shown of 5 replicates. (C) Non-parametric analyses of the non-normalised raw data revealed a statistically significant 2-fold increase in h-CaD phosphorylation during the transition from point 1 to point 2. No significant difference was observed between contraction points 1 and 3 or 2 and 3. *p

    Techniques Used: In Vitro, Protein Extraction, Polyacrylamide Gel Electrophoresis

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    Immunocytochemistry:

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    Immunostaining:

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    Schematic representation of the functions of µ- and m-calpain in LTP induction and consolidation a . µ-calpain activation is necessary for synaptic potentiation during LTP induction, and its inhibition prevents LTP (middle panel). m-calpain activation during consolidation limits the extent of synaptic potentiation and its inhibition results in enhanced LTP (right panel). µ-calpain activation is indicated by yellow triangles and m-calpain by red squares. Calpain inhibitor III (CI–III) application is indicated by red arrows. Note that we also postulate that µ-calpain and m-calpain are differentially localized in synapses. b . Signaling pathways downstream of µ- and m- calpain in LTP induction and consolidation. In LTP induction, µ-calpain activation, possibly resulting from Ca 2+ influx through the NMDA receptors, results in <t>SCOP</t> truncation followed by <t>ERK</t> activation. In the consolidation period, m-calpain activation, possibly resulting from BDNF-mediated ERK activation 13 stimulates mTOR-dependent protein synthesis through calpain-mediated PTEN degradation 14 , and in particular SCOP synthesis, which would restore normal SCOP levels, thereby preventing ERK activation.
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    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
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    A New Live Sensor of ERK (A) Schematic of the miniCic construct. Right cartoon: expected localization of miniCic at low ERK activity (accumulation in nucleus, n) and high ERK activity (exclusion in the cytoplasm, c) is shown. (B) z-projection of immunostainings of a wing imaginal disc (top) and an eye imaginal disc (bottom) at the L3 wandering stage (10/10 tissues for each) stained for DAPI (blue), E-cad (white), <t>dpERK</t> (green), and miniCic (anti-mCherry, red; purple on the overlay). Scale bars represent 10 μm. Close-up views (dashed white rectangles) are shown on the right (single plane; dpERK pseudocolor; miniCic greyscale); graphs: intensity line profiles of dpERK (green) and miniCic (purple) along white dashed rectangles. (C) Live pupal nota expressing miniCic and <t>endo-Ecad::GFP</t> with EGFR-depleted clones (left, green GFP; UAS-egfr dsRNA ) or clones overexpressing hEGFR (right, UAS-hegfr::GFP ). Top schemes: localization of the clones in the notum is shown (red rectangles; A, anterior; P, posterior; dashed lines, midline; zone of high ERK activity on the left and low ERK activity for the right). Blue arrows: ectopic nuclear accumulation. White dashed lines: clone boundaries. Scale bars represent 10 μm. (D) Western blot of dpERK (top) and total ERK (bottom) in S2 cells upon inhibition of ERK phosphorylation by trametinib (10 μM; a potent inhibitor of MEK, left scheme) at different time after drug treatment (in minutes). Control band: DMSO treatment (30 min after adding DMSO). Similar results were obtained at 1 μM. (E) Live imaging of larval haemocytes primary culture expressing tub-miniCic upon treatment with 10 μM trametinib (top) or DMSO (bottom). Time (minutes) after drug deposition is shown. Dotted circles: nuclei (detected by transmitted light). Scale bars represent 10 μm. (Right graph) Mean normalized miniCic nuclear intensity after drug treatment (two independent experiments) is shown. Error bars are SEM. (F) Snapshots of a live pupal notum expressing endo-Ecad::GFP and miniCic (local z-projection) at different time after pupal formation (APF). Anterior: left; posterior: right; midline in the center (see left scheme). Scale bar represents 20 μm. Right heatmap: averaged miniCic signal over the full movie is shown; cell death events over the full movie are shown (bottom right picture; one dot = one elimination). .
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    Activation of downstream targets of the MAPK pathway within 13-16 days after TAM treatment. A) Western blot for phospho MEK, phospho Erk1/2, and phosphor <t>p38</t> in K-ras activated mice and control mice. Immunohistochemical staining for phospho Erk1/2 in the stomach of the B) control and C) K-ras activated mice, or for phospho p38 ( D and E ), respectively, 15 days after tamoxifen feeding. Scale bar 200μM.
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    Schematic representation of the functions of µ- and m-calpain in LTP induction and consolidation a . µ-calpain activation is necessary for synaptic potentiation during LTP induction, and its inhibition prevents LTP (middle panel). m-calpain activation during consolidation limits the extent of synaptic potentiation and its inhibition results in enhanced LTP (right panel). µ-calpain activation is indicated by yellow triangles and m-calpain by red squares. Calpain inhibitor III (CI–III) application is indicated by red arrows. Note that we also postulate that µ-calpain and m-calpain are differentially localized in synapses. b . Signaling pathways downstream of µ- and m- calpain in LTP induction and consolidation. In LTP induction, µ-calpain activation, possibly resulting from Ca 2+ influx through the NMDA receptors, results in SCOP truncation followed by ERK activation. In the consolidation period, m-calpain activation, possibly resulting from BDNF-mediated ERK activation 13 stimulates mTOR-dependent protein synthesis through calpain-mediated PTEN degradation 14 , and in particular SCOP synthesis, which would restore normal SCOP levels, thereby preventing ERK activation.

    Journal: Nature communications

    Article Title: A molecular brake controls the magnitude of long-term potentiation

    doi: 10.1038/ncomms4051

    Figure Lengend Snippet: Schematic representation of the functions of µ- and m-calpain in LTP induction and consolidation a . µ-calpain activation is necessary for synaptic potentiation during LTP induction, and its inhibition prevents LTP (middle panel). m-calpain activation during consolidation limits the extent of synaptic potentiation and its inhibition results in enhanced LTP (right panel). µ-calpain activation is indicated by yellow triangles and m-calpain by red squares. Calpain inhibitor III (CI–III) application is indicated by red arrows. Note that we also postulate that µ-calpain and m-calpain are differentially localized in synapses. b . Signaling pathways downstream of µ- and m- calpain in LTP induction and consolidation. In LTP induction, µ-calpain activation, possibly resulting from Ca 2+ influx through the NMDA receptors, results in SCOP truncation followed by ERK activation. In the consolidation period, m-calpain activation, possibly resulting from BDNF-mediated ERK activation 13 stimulates mTOR-dependent protein synthesis through calpain-mediated PTEN degradation 14 , and in particular SCOP synthesis, which would restore normal SCOP levels, thereby preventing ERK activation.

    Article Snippet: The primary antibodies used were SCOP (1:1000, 07-1341, Millipore), actin (1:10000, MAB1501, Millipore), phospho-ERK (1:2000, 9101, Cell Signaling), ERK (1:3000, 9107), and Akt (1:1500, 2920).

    Techniques: Activation Assay, Inhibition

    Effects of cycloheximide and rapamycin on BDNF-induced changes in levels of SCOP and p-ERK a, d , Experimental design: hippocampal slices were treated with BDNF (100 ng/ml) for the indicated periods of time in the presence of cycloheximide (25 µM) or rapamycin (1 µM). b, e , Representative western blots for SCOP, Akt, p-ERK and ERK under various experimental conditions. c, f . Quantitative analysis of the levels of SCOP (normalized to the values of Akt) and p-ERK/ERK ratios under various experimental conditions. In all cases, results are means ± S.E.M. of 3 experiments. * p

    Journal: Nature communications

    Article Title: A molecular brake controls the magnitude of long-term potentiation

    doi: 10.1038/ncomms4051

    Figure Lengend Snippet: Effects of cycloheximide and rapamycin on BDNF-induced changes in levels of SCOP and p-ERK a, d , Experimental design: hippocampal slices were treated with BDNF (100 ng/ml) for the indicated periods of time in the presence of cycloheximide (25 µM) or rapamycin (1 µM). b, e , Representative western blots for SCOP, Akt, p-ERK and ERK under various experimental conditions. c, f . Quantitative analysis of the levels of SCOP (normalized to the values of Akt) and p-ERK/ERK ratios under various experimental conditions. In all cases, results are means ± S.E.M. of 3 experiments. * p

    Article Snippet: The primary antibodies used were SCOP (1:1000, 07-1341, Millipore), actin (1:10000, MAB1501, Millipore), phospho-ERK (1:2000, 9101, Cell Signaling), ERK (1:3000, 9107), and Akt (1:1500, 2920).

    Techniques: Western Blot

    Effects of calpain inhibition on TBS-induced changes in adult hippocampal CA1 mini-slices a, d, g . Experimental protocol: hippocampal CA1 mini-slices were prepared from adult rats and were stimulated by TBS in the absence or presence of calpain inhibitor III (10 µM), as indicated. b, e, h . Representative western blots for SCOP, Akt, p-ERK and ERK under various experimental conditions. c, f, i . Quantitative analysis of the levels of SCOP (normalized to the values of Akt) and p-ERK/ERK ratios under various experimental conditions. Results are means ± S.E.M. of 3–8 experiments. * p

    Journal: Nature communications

    Article Title: A molecular brake controls the magnitude of long-term potentiation

    doi: 10.1038/ncomms4051

    Figure Lengend Snippet: Effects of calpain inhibition on TBS-induced changes in adult hippocampal CA1 mini-slices a, d, g . Experimental protocol: hippocampal CA1 mini-slices were prepared from adult rats and were stimulated by TBS in the absence or presence of calpain inhibitor III (10 µM), as indicated. b, e, h . Representative western blots for SCOP, Akt, p-ERK and ERK under various experimental conditions. c, f, i . Quantitative analysis of the levels of SCOP (normalized to the values of Akt) and p-ERK/ERK ratios under various experimental conditions. Results are means ± S.E.M. of 3–8 experiments. * p

    Article Snippet: The primary antibodies used were SCOP (1:1000, 07-1341, Millipore), actin (1:10000, MAB1501, Millipore), phospho-ERK (1:2000, 9101, Cell Signaling), ERK (1:3000, 9107), and Akt (1:1500, 2920).

    Techniques: Inhibition, Western Blot

    Effects of calpain inhibitor III on BDNF-induced changes in levels of SCOP and p-ERK a, d, g. Experimental protocols: hippocampal slices were prepared from adult rats and were treated with BDNF (100 ng/ml) for the indicated periods of time in the absence or presence of calpain inhibitor III (10 µM), as indicated. b, e, h. Representative western blots for SCOP, Akt, p-ERK and ERK under various experimental conditions. c, f, i. Quantitative analysis of the levels of SCOP (normalized to the values of Akt) and p-ERK/ERK ratios under various experimental conditions. In all cases, results are means ± S.E.M. of 3 experiments. * p

    Journal: Nature communications

    Article Title: A molecular brake controls the magnitude of long-term potentiation

    doi: 10.1038/ncomms4051

    Figure Lengend Snippet: Effects of calpain inhibitor III on BDNF-induced changes in levels of SCOP and p-ERK a, d, g. Experimental protocols: hippocampal slices were prepared from adult rats and were treated with BDNF (100 ng/ml) for the indicated periods of time in the absence or presence of calpain inhibitor III (10 µM), as indicated. b, e, h. Representative western blots for SCOP, Akt, p-ERK and ERK under various experimental conditions. c, f, i. Quantitative analysis of the levels of SCOP (normalized to the values of Akt) and p-ERK/ERK ratios under various experimental conditions. In all cases, results are means ± S.E.M. of 3 experiments. * p

    Article Snippet: The primary antibodies used were SCOP (1:1000, 07-1341, Millipore), actin (1:10000, MAB1501, Millipore), phospho-ERK (1:2000, 9101, Cell Signaling), ERK (1:3000, 9107), and Akt (1:1500, 2920).

    Techniques: Western Blot

    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition. a and b Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activation Assay, Activity Assay, Inhibition, Western Blot, Cell Culture

    The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activity Assay, Functional Assay

    BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals.  a  Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126.  b  Quantification analysis of the infarct volume [n = 6, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals. a Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126. b Quantification analysis of the infarct volume [n = 6, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Inhibition, Activation Assay, Staining

    BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation.  a  Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation. a Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Functional Assay, Inhibition, Activation Assay, Injection

    After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased.  a  and  b  Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm.  c  and  d  Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased. a and b Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm. c and d Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Double Immunofluorescence Staining, Western Blot, Expressing

    BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury.  a  A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure.  b  and  c  Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury. a A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure. b and c Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Western Blot, Expressing

    BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level.  a  Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level. a Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activity Assay, Western Blot, Expressing

    A New Live Sensor of ERK (A) Schematic of the miniCic construct. Right cartoon: expected localization of miniCic at low ERK activity (accumulation in nucleus, n) and high ERK activity (exclusion in the cytoplasm, c) is shown. (B) z-projection of immunostainings of a wing imaginal disc (top) and an eye imaginal disc (bottom) at the L3 wandering stage (10/10 tissues for each) stained for DAPI (blue), E-cad (white), dpERK (green), and miniCic (anti-mCherry, red; purple on the overlay). Scale bars represent 10 μm. Close-up views (dashed white rectangles) are shown on the right (single plane; dpERK pseudocolor; miniCic greyscale); graphs: intensity line profiles of dpERK (green) and miniCic (purple) along white dashed rectangles. (C) Live pupal nota expressing miniCic and endo-Ecad::GFP with EGFR-depleted clones (left, green GFP; UAS-egfr dsRNA ) or clones overexpressing hEGFR (right, UAS-hegfr::GFP ). Top schemes: localization of the clones in the notum is shown (red rectangles; A, anterior; P, posterior; dashed lines, midline; zone of high ERK activity on the left and low ERK activity for the right). Blue arrows: ectopic nuclear accumulation. White dashed lines: clone boundaries. Scale bars represent 10 μm. (D) Western blot of dpERK (top) and total ERK (bottom) in S2 cells upon inhibition of ERK phosphorylation by trametinib (10 μM; a potent inhibitor of MEK, left scheme) at different time after drug treatment (in minutes). Control band: DMSO treatment (30 min after adding DMSO). Similar results were obtained at 1 μM. (E) Live imaging of larval haemocytes primary culture expressing tub-miniCic upon treatment with 10 μM trametinib (top) or DMSO (bottom). Time (minutes) after drug deposition is shown. Dotted circles: nuclei (detected by transmitted light). Scale bars represent 10 μm. (Right graph) Mean normalized miniCic nuclear intensity after drug treatment (two independent experiments) is shown. Error bars are SEM. (F) Snapshots of a live pupal notum expressing endo-Ecad::GFP and miniCic (local z-projection) at different time after pupal formation (APF). Anterior: left; posterior: right; midline in the center (see left scheme). Scale bar represents 20 μm. Right heatmap: averaged miniCic signal over the full movie is shown; cell death events over the full movie are shown (bottom right picture; one dot = one elimination). .

    Journal: Current Biology

    Article Title: Competition for Space Induces Cell Elimination through Compaction-Driven ERK Downregulation

    doi: 10.1016/j.cub.2018.11.007

    Figure Lengend Snippet: A New Live Sensor of ERK (A) Schematic of the miniCic construct. Right cartoon: expected localization of miniCic at low ERK activity (accumulation in nucleus, n) and high ERK activity (exclusion in the cytoplasm, c) is shown. (B) z-projection of immunostainings of a wing imaginal disc (top) and an eye imaginal disc (bottom) at the L3 wandering stage (10/10 tissues for each) stained for DAPI (blue), E-cad (white), dpERK (green), and miniCic (anti-mCherry, red; purple on the overlay). Scale bars represent 10 μm. Close-up views (dashed white rectangles) are shown on the right (single plane; dpERK pseudocolor; miniCic greyscale); graphs: intensity line profiles of dpERK (green) and miniCic (purple) along white dashed rectangles. (C) Live pupal nota expressing miniCic and endo-Ecad::GFP with EGFR-depleted clones (left, green GFP; UAS-egfr dsRNA ) or clones overexpressing hEGFR (right, UAS-hegfr::GFP ). Top schemes: localization of the clones in the notum is shown (red rectangles; A, anterior; P, posterior; dashed lines, midline; zone of high ERK activity on the left and low ERK activity for the right). Blue arrows: ectopic nuclear accumulation. White dashed lines: clone boundaries. Scale bars represent 10 μm. (D) Western blot of dpERK (top) and total ERK (bottom) in S2 cells upon inhibition of ERK phosphorylation by trametinib (10 μM; a potent inhibitor of MEK, left scheme) at different time after drug treatment (in minutes). Control band: DMSO treatment (30 min after adding DMSO). Similar results were obtained at 1 μM. (E) Live imaging of larval haemocytes primary culture expressing tub-miniCic upon treatment with 10 μM trametinib (top) or DMSO (bottom). Time (minutes) after drug deposition is shown. Dotted circles: nuclei (detected by transmitted light). Scale bars represent 10 μm. (Right graph) Mean normalized miniCic nuclear intensity after drug treatment (two independent experiments) is shown. Error bars are SEM. (F) Snapshots of a live pupal notum expressing endo-Ecad::GFP and miniCic (local z-projection) at different time after pupal formation (APF). Anterior: left; posterior: right; midline in the center (see left scheme). Scale bar represents 20 μm. Right heatmap: averaged miniCic signal over the full movie is shown; cell death events over the full movie are shown (bottom right picture; one dot = one elimination). .

    Article Snippet: The following antibodies/markers were used: rat anti E-cad (DCAD2 concentrate, DSHB, 1/50), guinea pig anti Hid (1/50, strepatavidin amplified, gift of Don Hyong Ryu), mouse anti EcR (DSHB Ag10.2 concentrated, 1/100), rabbit anti dpERK (Cell signaling, #4370, 1/100), chicken anti GFP (abCam, #13970, 1/500), chicken anti mCherry (abCam, #205402, 1/200), rabbit anti-cleaved Dcp-1 (Cell Signaling, #9578, 1/50), mouse anti-Argos (DSHB concentrated, 1/50), monoclonal mouse anti EGFR (Sigma, E29006 clone C-273, 1/50).

    Techniques: Construct, Activity Assay, Staining, Expressing, Clone Assay, Western Blot, Inhibition, Imaging

    Activation of downstream targets of the MAPK pathway within 13-16 days after TAM treatment. A) Western blot for phospho MEK, phospho Erk1/2, and phosphor p38 in K-ras activated mice and control mice. Immunohistochemical staining for phospho Erk1/2 in the stomach of the B) control and C) K-ras activated mice, or for phospho p38 ( D and E ), respectively, 15 days after tamoxifen feeding. Scale bar 200μM.

    Journal: American Journal of Cancer Research

    Article Title: Systemic activation of K-ras rapidly induces gastric hyperplasia and metaplasia in mice

    doi:

    Figure Lengend Snippet: Activation of downstream targets of the MAPK pathway within 13-16 days after TAM treatment. A) Western blot for phospho MEK, phospho Erk1/2, and phosphor p38 in K-ras activated mice and control mice. Immunohistochemical staining for phospho Erk1/2 in the stomach of the B) control and C) K-ras activated mice, or for phospho p38 ( D and E ), respectively, 15 days after tamoxifen feeding. Scale bar 200μM.

    Article Snippet: The primary antibodies used were Phospho Mek, Mek, phospho Erk1/2, Erk 1/2, phospho p38, p38 (1:1000, Cell Signaling Technologies), CD44, Lgr5 (1:1000 Abcam) and CDX2 (1:1000 Biogenex).

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