Structured Review

Cell Signaling Technology Inc phospho erk
Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and <t>ERK</t> or phospho-MET, <t>phospho-Akt</t> and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).
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1) Product Images from "A cysteine selenosulfide redox switch for protein chemical synthesis"

Article Title: A cysteine selenosulfide redox switch for protein chemical synthesis

Journal: Nature Communications

doi: 10.1038/s41467-020-16359-6

Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).
Figure Legend Snippet: Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).

Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Amplified Luminescent Proximity Homogenous Assay, Recombinant, Incubation, Standard Deviation, Phosphorylation Assay, Western Blot, Stripping Membranes, Scattering Assay, Isolation, Staining

2) Product Images from "Apigenin alleviates TGF-β1-induced nasal mucosa remodeling by inhibiting MAPK / NF-kB signaling pathways in chronic rhinosinusitis"

Article Title: Apigenin alleviates TGF-β1-induced nasal mucosa remodeling by inhibiting MAPK / NF-kB signaling pathways in chronic rhinosinusitis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201595

Effect of apigenin on the TGF-β1-induced MAPK signaling pathway in nasal fibroblasts. Nasal fibroblasts were pretreated with apigenin (5 μM) for 1 hour and combined with TGF-β1 (5 ng/ml). (A) Phosphorylation of MAPK (p-p38, p-JNK, and p-ERK) was detected by western blot. (B) Nasal fibroblasts were stimulated with TGF-β1 with or without the following specific inhibitors: SB203580 (10 μM), SP600125 (5 μM). Protein expression levels of a -SMA, fibronectin, and collagen type I were determined by western blot. (C) Total collagen was evaluated by sircol assay. Results were obtained from at least three independent experiments. * p
Figure Legend Snippet: Effect of apigenin on the TGF-β1-induced MAPK signaling pathway in nasal fibroblasts. Nasal fibroblasts were pretreated with apigenin (5 μM) for 1 hour and combined with TGF-β1 (5 ng/ml). (A) Phosphorylation of MAPK (p-p38, p-JNK, and p-ERK) was detected by western blot. (B) Nasal fibroblasts were stimulated with TGF-β1 with or without the following specific inhibitors: SB203580 (10 μM), SP600125 (5 μM). Protein expression levels of a -SMA, fibronectin, and collagen type I were determined by western blot. (C) Total collagen was evaluated by sircol assay. Results were obtained from at least three independent experiments. * p

Techniques Used: Western Blot, Expressing

3) Product Images from "Periplaneta americana Extracts Promote Skin Wound Healing via Nuclear Factor Kappa B Canonical Pathway and Extracellular Signal-Regulated Kinase Signaling"

Article Title: Periplaneta americana Extracts Promote Skin Wound Healing via Nuclear Factor Kappa B Canonical Pathway and Extracellular Signal-Regulated Kinase Signaling

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2017/5821706

Immunofluorescence analysis of the spatial localization of the NF- κ B canonical and ERK pathways. Cells were treated with PAE for 48 h, fixed, and incubated to determine the immunofluorescence. The antibodies against (a) RelA, (b) p-ERK, and (c) ERK presented green fluorescence, whereas a blue fluorescent signal was generated by DAPI staining of the cell nuclei. Areas of overlap between the green and the blue fluorescence appeared as merged images.
Figure Legend Snippet: Immunofluorescence analysis of the spatial localization of the NF- κ B canonical and ERK pathways. Cells were treated with PAE for 48 h, fixed, and incubated to determine the immunofluorescence. The antibodies against (a) RelA, (b) p-ERK, and (c) ERK presented green fluorescence, whereas a blue fluorescent signal was generated by DAPI staining of the cell nuclei. Areas of overlap between the green and the blue fluorescence appeared as merged images.

Techniques Used: Immunofluorescence, Incubation, Fluorescence, Generated, Staining

4) Product Images from "Sequential Delivery of Synaptic GluA1- and GluA4-containing AMPA Receptors (AMPARs) by SAP97 Anchored Protein Complexes in Classical Conditioning *"

Article Title: Sequential Delivery of Synaptic GluA1- and GluA4-containing AMPA Receptors (AMPARs) by SAP97 Anchored Protein Complexes in Classical Conditioning *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.535179

Model summarizing our findings on the role of SAP97 and other scaffolding proteins and kinases on subunit-specific synaptic delivery of AMPARs during early and later stages of in vitro classical conditioning. A , about 15 min after conditioning onset, a SAP97-AKAP/PKA-GluA1 protein complex forms that is initiated by the phosphorylation of PKA. B , shortly after A at about C1 (25 min), this complex translocates to the PSD where there is an interaction between SAP97-PSD95 and GluA1-containing AMPARs are released from SAP97 for delivery to the synapse. C , still later in conditioning at about C2 (80 min after conditioning onset), a SAP97-KSR1/PKC-GluA4 complex forms. This occurs while SAP97 is already bound to PSD95 and/or there are new SAP97 complexes that translocate to the PSD to deliver GluA4 AMPAR subunits to the synapse. This step requires the phosphorylation of ERK. During this phase GluA1-containing AMPARs are removed from the membrane surface.
Figure Legend Snippet: Model summarizing our findings on the role of SAP97 and other scaffolding proteins and kinases on subunit-specific synaptic delivery of AMPARs during early and later stages of in vitro classical conditioning. A , about 15 min after conditioning onset, a SAP97-AKAP/PKA-GluA1 protein complex forms that is initiated by the phosphorylation of PKA. B , shortly after A at about C1 (25 min), this complex translocates to the PSD where there is an interaction between SAP97-PSD95 and GluA1-containing AMPARs are released from SAP97 for delivery to the synapse. C , still later in conditioning at about C2 (80 min after conditioning onset), a SAP97-KSR1/PKC-GluA4 complex forms. This occurs while SAP97 is already bound to PSD95 and/or there are new SAP97 complexes that translocate to the PSD to deliver GluA4 AMPAR subunits to the synapse. This step requires the phosphorylation of ERK. During this phase GluA1-containing AMPARs are removed from the membrane surface.

Techniques Used: Scaffolding, In Vitro

5) Product Images from "Essential Role of the Redox Sensitive Kinase p66shc in Determining Energetic and Oxidative Status and Cell Fate in Neuronal Preconditioning"

Article Title: Essential Role of the Redox Sensitive Kinase p66shc in Determining Energetic and Oxidative Status and Cell Fate in Neuronal Preconditioning

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.6366-09.2010

Raf Activation is Required for Preconditioning Protection and Upregulation of HSP70 Inhibitors of Raf, p42/44 ERK or p38 were added during preconditioning as outlined in Experimental Methods. Twenty-four hours after preconditioning, cultures were washed and exposed to 100µM NMDA with 10µM glycine for 1h. A) Cell death was assessed 24h later by LDH release from dead and dying neurons. Data represent the mean ± SEM for five independent experiments, and were analyzed using one-way ANOVA. Posthoc analysis was done using Tukey test comparing groups to PC alone. Δ is used to denote statistical significance compared to preconditioning alone. Both asterisks and Δ represent p
Figure Legend Snippet: Raf Activation is Required for Preconditioning Protection and Upregulation of HSP70 Inhibitors of Raf, p42/44 ERK or p38 were added during preconditioning as outlined in Experimental Methods. Twenty-four hours after preconditioning, cultures were washed and exposed to 100µM NMDA with 10µM glycine for 1h. A) Cell death was assessed 24h later by LDH release from dead and dying neurons. Data represent the mean ± SEM for five independent experiments, and were analyzed using one-way ANOVA. Posthoc analysis was done using Tukey test comparing groups to PC alone. Δ is used to denote statistical significance compared to preconditioning alone. Both asterisks and Δ represent p

Techniques Used: Activation Assay

6) Product Images from "miR-1260b, mediated by YY1, activates KIT signaling by targeting SOCS6 to regulate cell proliferation and apoptosis in NSCLC"

Article Title: miR-1260b, mediated by YY1, activates KIT signaling by targeting SOCS6 to regulate cell proliferation and apoptosis in NSCLC

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1390-y

miR-1260b-mediated suppression of SOCS6 activated KIT signaling. Expression levels of SOCS6, KIT, p-p38, p-ERK, and ERK were detected by western blot. GAPDH was used as an internal control. 1: Lv-vector, 2: Lv-miR-1260b, 3: si-NC, 4: si-SOCS6 in SPCA1 cell line; 1: Sh-vector, 2: Sh-miR-1260b, 3: NC, 4: SOCS6 in H1299 cell line. The data expressed as the mean ± SD (* P
Figure Legend Snippet: miR-1260b-mediated suppression of SOCS6 activated KIT signaling. Expression levels of SOCS6, KIT, p-p38, p-ERK, and ERK were detected by western blot. GAPDH was used as an internal control. 1: Lv-vector, 2: Lv-miR-1260b, 3: si-NC, 4: si-SOCS6 in SPCA1 cell line; 1: Sh-vector, 2: Sh-miR-1260b, 3: NC, 4: SOCS6 in H1299 cell line. The data expressed as the mean ± SD (* P

Techniques Used: Expressing, Western Blot, Plasmid Preparation

7) Product Images from "NF-?B inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death"

Article Title: NF-?B inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg379

Fig. 1. Prolonged MAPK activation by TNF, but not IL-1, in DKO and p65KO MEFs. ( A , B , E and F ) Wild-type, DKO and p65KO MEFs were stimulated with TNF (50 ng/ml) for the indicated time periods, and then the lysates were blotted with antibodies specific for the activated form of JNK (phospho-JNK), p38 (phospho-p38) or ERK (phospho-ERK). The membranes were reblotted with antibodies to total JNK, p38 or ERK. ( C and D ) Wild-type, DKO and p65KO MEFs were stimulated with IL-1 (50 ng/ml) for the indicated time periods and the lysates were blotted as above.
Figure Legend Snippet: Fig. 1. Prolonged MAPK activation by TNF, but not IL-1, in DKO and p65KO MEFs. ( A , B , E and F ) Wild-type, DKO and p65KO MEFs were stimulated with TNF (50 ng/ml) for the indicated time periods, and then the lysates were blotted with antibodies specific for the activated form of JNK (phospho-JNK), p38 (phospho-p38) or ERK (phospho-ERK). The membranes were reblotted with antibodies to total JNK, p38 or ERK. ( C and D ) Wild-type, DKO and p65KO MEFs were stimulated with IL-1 (50 ng/ml) for the indicated time periods and the lysates were blotted as above.

Techniques Used: Activation Assay

8) Product Images from "Gomisin G Suppresses the Growth of Colon Cancer Cells by Attenuation of AKT Phosphorylation and Arrest of Cell Cycle Progression"

Article Title: Gomisin G Suppresses the Growth of Colon Cancer Cells by Attenuation of AKT Phosphorylation and Arrest of Cell Cycle Progression

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2018.054

Gomisin G mediated alteration of AKT phosphorylation in LoVo cells. LoVo cells were treated with 10 μM Gomisin G or Gomisin O for 24 h and lysed. Equal concentrations of proteins were separated by SDS-PAGE and transferred to membrane blots. The blots were detected with pAKT, AKT, pERK, ERK, pp38, and p38 antibodies. β-actin was used as a loading control.
Figure Legend Snippet: Gomisin G mediated alteration of AKT phosphorylation in LoVo cells. LoVo cells were treated with 10 μM Gomisin G or Gomisin O for 24 h and lysed. Equal concentrations of proteins were separated by SDS-PAGE and transferred to membrane blots. The blots were detected with pAKT, AKT, pERK, ERK, pp38, and p38 antibodies. β-actin was used as a loading control.

Techniques Used: SDS Page

9) Product Images from "Angiomotin-like Proteins Associate with and Negatively Regulate YAP1 *"

Article Title: Angiomotin-like Proteins Associate with and Negatively Regulate YAP1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.C110.205401

Down-regulation of AMOTL2 causes EMT in MCF10A cells. A , 293T cells were transfected with the indicated shRNAs together with plasmids encoding FLAG-tagged AMOTL1 or AMOTL2. Cells were collected 72 h later and subjected to Western blotting. Ctrl , control. B , the level of AMOTL1 or AMOTL2 transcripts was revealed by RT-PCR in the indicated stable knockdown cells. C , immunoprecipitation ( IP ) and immunoblotting were performed using anti-AMOTL2 serum and cell lysates prepared from the indicated cell lines. For each immunoprecipitation, a total of 1 mg of the indicated protein lysates was used. Anti-tubulin immunoblotting was included as a control. D , lentiviral shRNAs were used to infect MCF10A cells, and stable knockdown pools were generated. Bright field pictures were captured to reveal cell morphology in these pools. E , cells with AMOTL2 down-regulation displayed EMT phenotypes. E-cadherin was used as epithelial marker. N-cadherin and vimentin were used as mesenchymal markers. F , cell-cell junction was diminished in AMOTL2 knockdown cells. E-cadherin was used as cell-cell junction marker. Actin filaments were labeled by TRITC-phalloidin. M , merged. G , cell migration capability increased in AMOTL2 knockdown cells as determined by wound healing assay. H , MCF10A cells were infected with the indicated lentiviral shRNAs respectively, and stable pools were used for immunostaining with anti-E-cadherin and anti-vimentin antibodies. The efficiency of YAP1 down-regulation by shRNAs was verified by anti-YAP1 immunoblotting. I , YAP1 retained its dominant nuclear localization in AMOTL2 knockdown ( Ri ) cells even when cells reached confluence. M , merged. J , YAP1 phosphorylation ( p-YAP1 ) decreased in AMOTL2 knockdown MCF10A cells. AKT and ERK signaling pathways were also activated in AMOTL2 knockdown cells. p-AKT , AKT phosphorylation; p-ERK1/2 , ERK1/2 phosphorylation.
Figure Legend Snippet: Down-regulation of AMOTL2 causes EMT in MCF10A cells. A , 293T cells were transfected with the indicated shRNAs together with plasmids encoding FLAG-tagged AMOTL1 or AMOTL2. Cells were collected 72 h later and subjected to Western blotting. Ctrl , control. B , the level of AMOTL1 or AMOTL2 transcripts was revealed by RT-PCR in the indicated stable knockdown cells. C , immunoprecipitation ( IP ) and immunoblotting were performed using anti-AMOTL2 serum and cell lysates prepared from the indicated cell lines. For each immunoprecipitation, a total of 1 mg of the indicated protein lysates was used. Anti-tubulin immunoblotting was included as a control. D , lentiviral shRNAs were used to infect MCF10A cells, and stable knockdown pools were generated. Bright field pictures were captured to reveal cell morphology in these pools. E , cells with AMOTL2 down-regulation displayed EMT phenotypes. E-cadherin was used as epithelial marker. N-cadherin and vimentin were used as mesenchymal markers. F , cell-cell junction was diminished in AMOTL2 knockdown cells. E-cadherin was used as cell-cell junction marker. Actin filaments were labeled by TRITC-phalloidin. M , merged. G , cell migration capability increased in AMOTL2 knockdown cells as determined by wound healing assay. H , MCF10A cells were infected with the indicated lentiviral shRNAs respectively, and stable pools were used for immunostaining with anti-E-cadherin and anti-vimentin antibodies. The efficiency of YAP1 down-regulation by shRNAs was verified by anti-YAP1 immunoblotting. I , YAP1 retained its dominant nuclear localization in AMOTL2 knockdown ( Ri ) cells even when cells reached confluence. M , merged. J , YAP1 phosphorylation ( p-YAP1 ) decreased in AMOTL2 knockdown MCF10A cells. AKT and ERK signaling pathways were also activated in AMOTL2 knockdown cells. p-AKT , AKT phosphorylation; p-ERK1/2 , ERK1/2 phosphorylation.

Techniques Used: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Generated, Marker, Labeling, Migration, Wound Healing Assay, Infection, Immunostaining

10) Product Images from "Dual inhibition of EGFR and mTOR pathways in small cell lung cancer"

Article Title: Dual inhibition of EGFR and mTOR pathways in small cell lung cancer

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6605761

Effects on EGFR and mTOR pathways after treatment of SCLC cells with erlotinib, RAD001 and a combination of both. ( A ) GLC-4 and VL-68 cells do express EGFR. ( B ) GCL-4 and VL-68 cells were treated with 5 μ erlotinib±EGF (100 ng ml –1 ) for 10 min and blotted for p-ERK, p-AKT and respective total proteins. ( C ) VL-68 and GCL-4 cells were treated with 5 μ erlotinib, 5 n RAD001 or a combination of both for 24 h, and then immunoblotted for total and phospho-protein expression of AKT, ERK, mTOR and p70s6K.
Figure Legend Snippet: Effects on EGFR and mTOR pathways after treatment of SCLC cells with erlotinib, RAD001 and a combination of both. ( A ) GLC-4 and VL-68 cells do express EGFR. ( B ) GCL-4 and VL-68 cells were treated with 5 μ erlotinib±EGF (100 ng ml –1 ) for 10 min and blotted for p-ERK, p-AKT and respective total proteins. ( C ) VL-68 and GCL-4 cells were treated with 5 μ erlotinib, 5 n RAD001 or a combination of both for 24 h, and then immunoblotted for total and phospho-protein expression of AKT, ERK, mTOR and p70s6K.

Techniques Used: Gas Chromatography, Expressing

Immunostaining of EGFR and mTOR pathways in SCLC. Immunohistochemical staining of SCLC for ( A ) p-mTOR, ( B ) p-p70s6K (strongly stained mitoses are marked by arrows), ( C ) p-AKT, ( D ) p-ERK and ( E ) EGFR (all magnification × 400).
Figure Legend Snippet: Immunostaining of EGFR and mTOR pathways in SCLC. Immunohistochemical staining of SCLC for ( A ) p-mTOR, ( B ) p-p70s6K (strongly stained mitoses are marked by arrows), ( C ) p-AKT, ( D ) p-ERK and ( E ) EGFR (all magnification × 400).

Techniques Used: Immunostaining, Immunohistochemistry, Staining

11) Product Images from "Hypoxic preconditioning protection is eliminated in HIF-1α knockout mice subjected to neonatal hypoxia-ischemia"

Article Title: Hypoxic preconditioning protection is eliminated in HIF-1α knockout mice subjected to neonatal hypoxia-ischemia

Journal: Pediatric research

doi: 10.1038/pr.2014.53

ERK activation. Bar graph summarizes the ratio of pERK to ERK compared to wildtype naïve, there were no significant changes, ( a ) After hypoxia. There was a trend toward an increase in the wildtype cortex at 15 min (p=0.09). ( b ) After HI. There were trends toward an increase in HIFKO cortex at 15 min (p
Figure Legend Snippet: ERK activation. Bar graph summarizes the ratio of pERK to ERK compared to wildtype naïve, there were no significant changes, ( a ) After hypoxia. There was a trend toward an increase in the wildtype cortex at 15 min (p=0.09). ( b ) After HI. There were trends toward an increase in HIFKO cortex at 15 min (p

Techniques Used: Activation Assay

12) Product Images from "Tanshinone-1 induces tumor cell killing, enhanced by inhibition of secondary activation of signaling networks"

Article Title: Tanshinone-1 induces tumor cell killing, enhanced by inhibition of secondary activation of signaling networks

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.443

Tanshinone-1 activated p38, AKT, and ERK signaling. ( a ) Signaling pathways consisting of Stat3, p38, AKT, and ERK, may crosstalk and influence their respective biological effects. ( b and c ) Phosphorylation was analyzed by western blotting. Tanshinone-1 (40 μ M, Tan-1) enhanced phosphorylation of p38, AKT, and ERK in MDR KB/VCR and parental KB cells ( b ) and in MDR K562/A02 and parental K562 cells ( c ). ( d ) The impact of p38, AKT, and ERK inhibitors on Tan-1-mediated enhancement of cellular p38, AKT, and ERK phosphorylation. KB/VCR cells were pretreated with PI103 (10 μ M, AKT) or AZD6244 (5 μ M, ERK) for 30 min or SB203580 (50 μ M, p38) for 10 min. The cells were then exposed to 20 μ M of Tan-1 for 15 min (p-p38 and p-ERK) or 4 h (p-AKT and p-705-Stat3) followed by western blotting analyses. ( e ) The impact of siStat3-induced Stat3 reduction on the Tan-1-mediated phosphorylation of p38, AKT, and ERK. KB/VCR cells were pretreated with siCtrl or siStat3 for 48 h. The cells were then exposed to 40 μ M of Tan-1 for the indicated time and were analyzed by western blotting
Figure Legend Snippet: Tanshinone-1 activated p38, AKT, and ERK signaling. ( a ) Signaling pathways consisting of Stat3, p38, AKT, and ERK, may crosstalk and influence their respective biological effects. ( b and c ) Phosphorylation was analyzed by western blotting. Tanshinone-1 (40 μ M, Tan-1) enhanced phosphorylation of p38, AKT, and ERK in MDR KB/VCR and parental KB cells ( b ) and in MDR K562/A02 and parental K562 cells ( c ). ( d ) The impact of p38, AKT, and ERK inhibitors on Tan-1-mediated enhancement of cellular p38, AKT, and ERK phosphorylation. KB/VCR cells were pretreated with PI103 (10 μ M, AKT) or AZD6244 (5 μ M, ERK) for 30 min or SB203580 (50 μ M, p38) for 10 min. The cells were then exposed to 20 μ M of Tan-1 for 15 min (p-p38 and p-ERK) or 4 h (p-AKT and p-705-Stat3) followed by western blotting analyses. ( e ) The impact of siStat3-induced Stat3 reduction on the Tan-1-mediated phosphorylation of p38, AKT, and ERK. KB/VCR cells were pretreated with siCtrl or siStat3 for 48 h. The cells were then exposed to 40 μ M of Tan-1 for the indicated time and were analyzed by western blotting

Techniques Used: Western Blot

Cotreatments of p38, AKT and ERK inhibitors potentiated the tanshinone-1 (Tan-1)-induced apoptosis and cytotoxicity. ( a – c ) MDR KB/VCR and parental KB cells were pretreated with PI103 (10 μ M, AKT) or AZD6244 (5 μ M, ERK) for 30 min or SB203580 (50 μ M, p38) for 10 min. The cells were then exposed to 20 μ M of Tan-1 for 14 h. The cleavage of procaspase3 and PARP were detected by western blotting ( a ) and apoptotic induction was examined by flow cytometry ( b ). The apoptosis percentage from three independent experiments was expressed as mean±S.D.; * P
Figure Legend Snippet: Cotreatments of p38, AKT and ERK inhibitors potentiated the tanshinone-1 (Tan-1)-induced apoptosis and cytotoxicity. ( a – c ) MDR KB/VCR and parental KB cells were pretreated with PI103 (10 μ M, AKT) or AZD6244 (5 μ M, ERK) for 30 min or SB203580 (50 μ M, p38) for 10 min. The cells were then exposed to 20 μ M of Tan-1 for 14 h. The cleavage of procaspase3 and PARP were detected by western blotting ( a ) and apoptotic induction was examined by flow cytometry ( b ). The apoptosis percentage from three independent experiments was expressed as mean±S.D.; * P

Techniques Used: Western Blot, Flow Cytometry, Cytometry

13) Product Images from "Identification of pY19-caveolin-2 as a positive regulator of insulin-stimulated actin cytoskeleton-dependent mitogenesis"

Article Title: Identification of pY19-caveolin-2 as a positive regulator of insulin-stimulated actin cytoskeleton-dependent mitogenesis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2008.00391.x

Effect of cytochalasin-D and latrunculin B on insulin-induced nuclear co-localization of pY19-caveolin - 2 with phospho-ERK. Confluent Hirc-B cells grown on coverslips were left untreated (C) ( a ), challenged with 100 nM insulin for 10 min. (I) ( b ), treated with 1 μM CCD for 15 min. alone (CCD) ( c ), pre-treated with 1 μM CCD for 15 min. before 100 nM insulin stimulation for 10 min. (CCD+I) ( d ), treated with 1 μM LatB for 30 min. alone (LatB) ( e ), pretreated with 1 μM LatB for 30 min. before 100 nM insulin stimulation for 10 min. (LatB+I) ( f ). Cells were then fixed, permeabilized and stained with anti-F-actin, anti-pY19-caveolin-2 and anti-phospho-ERK antibodies followed by TRITC- or Alexa Fluor® 488-conjugated antibodies as described under ‘Materials and Methods’. DNA was stained using DAPI to visualize the nucleus and F-actin using FITC-conju-gated phalloidin. Coverslips were mounted on a slide and analysed by fluorescence microscopy. Gray : F-actin, Red : pY19-caveolin-2, Green : phospho-ERK, Merge : pY19-caveolin-2 + phospho-ERK + nucleus (DAPI). Co-localization of pY19-caveolin-2 with phospho-ERK to the nucleus was quantified by ImageJ. Percentage of nuclear co-localization from nuclear:cytoplasmic ratios of pY19-caveolin-2 with phospho-ERK staining is shown. The results represent mean ± S.E. from analysis of five separate field images of three independent experiments. ( C ) Hirc-B cells were treated with CCD (1 μM) for 15 min. or LatB (1 μM) for 30 min. before insulin treatment (100 nM) for 10 min. and subjected to nuclear frac-tionation using nuclear extraction kit as described under ‘Materials and Methods’. The cytoplasmic and nuclear fractions were analysed by immunoblot-ting using antibodies specific for phospho-ERK, pY19- caveolin-2, α-tubu-lin, and ku-70. Quantification shown represents the relative levels of phospho-ERK cellular distribution (%) in cytoplasm and nucleus detected as compared to the control sample; mean ± S.E., n = 3.
Figure Legend Snippet: Effect of cytochalasin-D and latrunculin B on insulin-induced nuclear co-localization of pY19-caveolin - 2 with phospho-ERK. Confluent Hirc-B cells grown on coverslips were left untreated (C) ( a ), challenged with 100 nM insulin for 10 min. (I) ( b ), treated with 1 μM CCD for 15 min. alone (CCD) ( c ), pre-treated with 1 μM CCD for 15 min. before 100 nM insulin stimulation for 10 min. (CCD+I) ( d ), treated with 1 μM LatB for 30 min. alone (LatB) ( e ), pretreated with 1 μM LatB for 30 min. before 100 nM insulin stimulation for 10 min. (LatB+I) ( f ). Cells were then fixed, permeabilized and stained with anti-F-actin, anti-pY19-caveolin-2 and anti-phospho-ERK antibodies followed by TRITC- or Alexa Fluor® 488-conjugated antibodies as described under ‘Materials and Methods’. DNA was stained using DAPI to visualize the nucleus and F-actin using FITC-conju-gated phalloidin. Coverslips were mounted on a slide and analysed by fluorescence microscopy. Gray : F-actin, Red : pY19-caveolin-2, Green : phospho-ERK, Merge : pY19-caveolin-2 + phospho-ERK + nucleus (DAPI). Co-localization of pY19-caveolin-2 with phospho-ERK to the nucleus was quantified by ImageJ. Percentage of nuclear co-localization from nuclear:cytoplasmic ratios of pY19-caveolin-2 with phospho-ERK staining is shown. The results represent mean ± S.E. from analysis of five separate field images of three independent experiments. ( C ) Hirc-B cells were treated with CCD (1 μM) for 15 min. or LatB (1 μM) for 30 min. before insulin treatment (100 nM) for 10 min. and subjected to nuclear frac-tionation using nuclear extraction kit as described under ‘Materials and Methods’. The cytoplasmic and nuclear fractions were analysed by immunoblot-ting using antibodies specific for phospho-ERK, pY19- caveolin-2, α-tubu-lin, and ku-70. Quantification shown represents the relative levels of phospho-ERK cellular distribution (%) in cytoplasm and nucleus detected as compared to the control sample; mean ± S.E., n = 3.

Techniques Used: Staining, Fluorescence, Microscopy

Effects of U0126 and wortmannin on insulin-induced nuclear co-localization of pY19-caveolin-2 with phospho-ERK. ( A ) Confluent Hirc-B fibrob-last monolayers grown on coverslips were left untreated (C); ( a ), or challenged for 10 min. with 100 nM insulin in the absence (I); ( b ) and presence of 10 μM U0126 for 2 hrs (UI); ( c ). Cells were then fixed, permeabilized and stained with anti-caveolin-2 antibody followed by TRITC-conjugated antibody and anti-ERK antibody followed by FITC-conjugated antibody as described under ‘Materials and Methods’. DNA was stained using DAPI to visualize the nucleus. Coverslips were mounted on a slide and analysed by fluorescence microscopy. Red : Caveolin-2, Green : ERK, Blue : Nucleus (DAPI), Merge1 : Caveolin-2 + ERK, and Merge2 : Caveolin-2 + ERK + DAPI. Co-localization of caveolin-2/ERK to the nucleus was quantified by ImageJ as described in ‘Materials and Methods’. Percentage of nuclear co-localization from nuclear:cytoplasmic ratios of caveolin-2 with ERK staining is shown. The results represent mean ± S.E. from analysis of five separate field images of five independent experiments. A minimum of 150 cells per condition were counted. ( B ) Cells were treated as follows: ( a ), control (C); ( b ), 100 nM insulin for 10 min. (I); ( c ), 10 μM U0126 for 2 hrs plus 100 nM insulin for 10 min. (UI); ( d ), 100 nM wortmannin for 1 hr plus 100 nM insulin for 10 min. (WI). After fixation and permeabilization, cells were stained with anti-pY19-caveolin-2 antibody followed by TRITC-conjugated antibody. DNA was stained using DAPI to visualize the nucleus. Red : pY19-Caveolin-2, Blue : Nucleus (DAPI), and Merge : pY19-Caveolin-2 + DAPI. pY19-Caveolin-2 nuclear translocation was quantified by ImageJ. Nuclear:cytoplasmic ratios of pY19-caveolin-2 staining is shown. The results represent mean ± S.E. from analysis of three separate field images of three independent experiments. ( C ) Cells were treated with or without U0126 (10 μM) for 2 hrs or with or without wortmannin (100 nM) for 1 hr before insulin treatment (100 nM) for 10 min. and subjected to nuclear fractionation using nuclear extraction kit as described under ‘Materials and Methods’. The cytoplasmic and nuclear fractions were analysed by immunoblotting using antibodies specific for phospho-ERK, pY19-caveolin-2, α-tubulin, and ku-70.
Figure Legend Snippet: Effects of U0126 and wortmannin on insulin-induced nuclear co-localization of pY19-caveolin-2 with phospho-ERK. ( A ) Confluent Hirc-B fibrob-last monolayers grown on coverslips were left untreated (C); ( a ), or challenged for 10 min. with 100 nM insulin in the absence (I); ( b ) and presence of 10 μM U0126 for 2 hrs (UI); ( c ). Cells were then fixed, permeabilized and stained with anti-caveolin-2 antibody followed by TRITC-conjugated antibody and anti-ERK antibody followed by FITC-conjugated antibody as described under ‘Materials and Methods’. DNA was stained using DAPI to visualize the nucleus. Coverslips were mounted on a slide and analysed by fluorescence microscopy. Red : Caveolin-2, Green : ERK, Blue : Nucleus (DAPI), Merge1 : Caveolin-2 + ERK, and Merge2 : Caveolin-2 + ERK + DAPI. Co-localization of caveolin-2/ERK to the nucleus was quantified by ImageJ as described in ‘Materials and Methods’. Percentage of nuclear co-localization from nuclear:cytoplasmic ratios of caveolin-2 with ERK staining is shown. The results represent mean ± S.E. from analysis of five separate field images of five independent experiments. A minimum of 150 cells per condition were counted. ( B ) Cells were treated as follows: ( a ), control (C); ( b ), 100 nM insulin for 10 min. (I); ( c ), 10 μM U0126 for 2 hrs plus 100 nM insulin for 10 min. (UI); ( d ), 100 nM wortmannin for 1 hr plus 100 nM insulin for 10 min. (WI). After fixation and permeabilization, cells were stained with anti-pY19-caveolin-2 antibody followed by TRITC-conjugated antibody. DNA was stained using DAPI to visualize the nucleus. Red : pY19-Caveolin-2, Blue : Nucleus (DAPI), and Merge : pY19-Caveolin-2 + DAPI. pY19-Caveolin-2 nuclear translocation was quantified by ImageJ. Nuclear:cytoplasmic ratios of pY19-caveolin-2 staining is shown. The results represent mean ± S.E. from analysis of three separate field images of three independent experiments. ( C ) Cells were treated with or without U0126 (10 μM) for 2 hrs or with or without wortmannin (100 nM) for 1 hr before insulin treatment (100 nM) for 10 min. and subjected to nuclear fractionation using nuclear extraction kit as described under ‘Materials and Methods’. The cytoplasmic and nuclear fractions were analysed by immunoblotting using antibodies specific for phospho-ERK, pY19-caveolin-2, α-tubulin, and ku-70.

Techniques Used: Staining, Fluorescence, Microscopy, Translocation Assay, Fractionation

14) Product Images from "CIN85 drives B cell responses by linking BCR signals to the canonical NF-?B pathway"

Article Title: CIN85 drives B cell responses by linking BCR signals to the canonical NF-?B pathway

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20102665

BCR-dependent activation of the canonical NF-κB pathway is impaired in CIN85 bKO B cells. (A) Purified spleen B cells were stimulated with 10 µg/ml anti-IgM F(ab’) 2 for indicated times (minutes). The cells were lysed and subjected to SDS-PAGE. Transferred membranes were probed with the indicated antibodies. (B) Spleen B cells from control or CIN85 bKO mice were stimulated with 10 µg/ml anti-IgM F(ab’) 2 fragment for the indicated times. After stimulation, some of the cells were subjected to SDS-PAGE to measure the phosphorylation status of IκBα (top). Other cells were collected, precipitated by anti–IKK-γ antibody and subjected to an IKK kinase assay using GST-IκBα as a substrate. The amount of phosphorylated GST-IκBα was detected by Western blotting as described in the Materials and methods (bottom). Phosphorylation status of IKK-β was also analyzed by Western blotting. p-ERK was examined with whole cell lysate. Representative data of at least three independent experiments are shown.
Figure Legend Snippet: BCR-dependent activation of the canonical NF-κB pathway is impaired in CIN85 bKO B cells. (A) Purified spleen B cells were stimulated with 10 µg/ml anti-IgM F(ab’) 2 for indicated times (minutes). The cells were lysed and subjected to SDS-PAGE. Transferred membranes were probed with the indicated antibodies. (B) Spleen B cells from control or CIN85 bKO mice were stimulated with 10 µg/ml anti-IgM F(ab’) 2 fragment for the indicated times. After stimulation, some of the cells were subjected to SDS-PAGE to measure the phosphorylation status of IκBα (top). Other cells were collected, precipitated by anti–IKK-γ antibody and subjected to an IKK kinase assay using GST-IκBα as a substrate. The amount of phosphorylated GST-IκBα was detected by Western blotting as described in the Materials and methods (bottom). Phosphorylation status of IKK-β was also analyzed by Western blotting. p-ERK was examined with whole cell lysate. Representative data of at least three independent experiments are shown.

Techniques Used: Activation Assay, Purification, SDS Page, Mouse Assay, Kinase Assay, Western Blot

15) Product Images from "Distinct Contributions of JNK and p38 to Chromium Cytotoxicity and Inhibition of Murine Embryonic Stem Cell Differentiation"

Article Title: Distinct Contributions of JNK and p38 to Chromium Cytotoxicity and Inhibition of Murine Embryonic Stem Cell Differentiation

Journal: Environmental Health Perspectives

doi: 10.1289/ehp.0800157

Distinct contributions of JNK and p38 to Cr(VI) toxicity. Cr(VI) induced the activation of the MAPKs via multiple mechanisms that can be ROS dependent and independent. The activation of JNK and p38, but not ERK, is mediated through MAP2K4 and MAP2K7. Specifically, MAP2K4 and MAP2K7 both are required for optimal JNK activation, but only MAP2K4 is essential for p38 activation. Using cells deficient in MAP2K4 and MAP2K7, we were able to delineate the distinct roles JNK and p38 play in the cytotoxicity and developmental toxicity of chromium.
Figure Legend Snippet: Distinct contributions of JNK and p38 to Cr(VI) toxicity. Cr(VI) induced the activation of the MAPKs via multiple mechanisms that can be ROS dependent and independent. The activation of JNK and p38, but not ERK, is mediated through MAP2K4 and MAP2K7. Specifically, MAP2K4 and MAP2K7 both are required for optimal JNK activation, but only MAP2K4 is essential for p38 activation. Using cells deficient in MAP2K4 and MAP2K7, we were able to delineate the distinct roles JNK and p38 play in the cytotoxicity and developmental toxicity of chromium.

Techniques Used: Activation Assay

MAP2K7 and JNK protect cells from cytotoxicity from high-concentration Cr(VI). WT, Map2k4 (−/−) , and Map2k7 (−/−) ES cells were treated with 50 μM Cr(VI) for different times as indicated. ( A ) MTS assay was performed to evaluate cell survival. Cell lysates were subjected to Western blotting to monitor the levels of apoptotic markers; ( B ) PARP cleavage and ( C ) active caspase-3. Map2k7 (−/−) cells were infected with adenoviruses for either GFP or MAP2K7 at 100 pfu/cell. At 48 hr of infection, ( D ) the expression of MAP2K7, GFP, and actin in WT, Map2k7 (−/−) , and Map2k7 (−/−) adenoviral-infected cells were examined by Western blotting. ( E ) WT, Map2k7 (−/−) , and Map2k7 (−/−) adenoviral-infected cells were treated with 50 μM Cr(VI) for 10 hr. Cell survival was determined by MTS assays, and each value was presented as mean ± SD from four replicates. ( F ) WT ES cells were pretreated with chemical inhibitor for JNK (SP60015, 5 μM), p38 (SB202190, 5 μM), ERK (PD98059, 5 μM), and protein synthesis (CHX, 1 μg/mL) for 1 hr. The cells were incubated with Cr(VI) at 50 μM for 8 hr. Cell survival rates were analyzed by MTS assay, and each value was presented as mean ± SD from four replicates. ** p
Figure Legend Snippet: MAP2K7 and JNK protect cells from cytotoxicity from high-concentration Cr(VI). WT, Map2k4 (−/−) , and Map2k7 (−/−) ES cells were treated with 50 μM Cr(VI) for different times as indicated. ( A ) MTS assay was performed to evaluate cell survival. Cell lysates were subjected to Western blotting to monitor the levels of apoptotic markers; ( B ) PARP cleavage and ( C ) active caspase-3. Map2k7 (−/−) cells were infected with adenoviruses for either GFP or MAP2K7 at 100 pfu/cell. At 48 hr of infection, ( D ) the expression of MAP2K7, GFP, and actin in WT, Map2k7 (−/−) , and Map2k7 (−/−) adenoviral-infected cells were examined by Western blotting. ( E ) WT, Map2k7 (−/−) , and Map2k7 (−/−) adenoviral-infected cells were treated with 50 μM Cr(VI) for 10 hr. Cell survival was determined by MTS assays, and each value was presented as mean ± SD from four replicates. ( F ) WT ES cells were pretreated with chemical inhibitor for JNK (SP60015, 5 μM), p38 (SB202190, 5 μM), ERK (PD98059, 5 μM), and protein synthesis (CHX, 1 μg/mL) for 1 hr. The cells were incubated with Cr(VI) at 50 μM for 8 hr. Cell survival rates were analyzed by MTS assay, and each value was presented as mean ± SD from four replicates. ** p

Techniques Used: Concentration Assay, MTS Assay, Western Blot, Infection, Expressing, Incubation

High-concentration Cr(VI) induces MAPK activation in WT, Map2k4 (−/−) , and Map2k7 (−/−) mouse ES cells. WT, Map2k4 (−/−) , and Map2k7 (−/−) cells were treated with 50 μM Cr(VI) for various times as indicated. Cell lysates were subjected to Western blot analysis for ( A ) phospho-JNK and total JNK, p38, and ERK; ( B ) phospho-c-JUN, ELK1, and ATF2; ( C ) total MAP2K4, MAP2K7, and actin; and ( D ) total c-JUN and c-FOS. Arrowheads point to specific MAPK isoforms. The ratio of phosphoprotein versus total protein or actin was calculated in each sample and the fold induction was calculated in comparison with the untreated control of the same cell type. Images shown in this figure are representative of at least two independent results.
Figure Legend Snippet: High-concentration Cr(VI) induces MAPK activation in WT, Map2k4 (−/−) , and Map2k7 (−/−) mouse ES cells. WT, Map2k4 (−/−) , and Map2k7 (−/−) cells were treated with 50 μM Cr(VI) for various times as indicated. Cell lysates were subjected to Western blot analysis for ( A ) phospho-JNK and total JNK, p38, and ERK; ( B ) phospho-c-JUN, ELK1, and ATF2; ( C ) total MAP2K4, MAP2K7, and actin; and ( D ) total c-JUN and c-FOS. Arrowheads point to specific MAPK isoforms. The ratio of phosphoprotein versus total protein or actin was calculated in each sample and the fold induction was calculated in comparison with the untreated control of the same cell type. Images shown in this figure are representative of at least two independent results.

Techniques Used: Concentration Assay, Activation Assay, Western Blot

NAC suppresses MAPK activation and cytotoxicity induced by high-concentration Cr(VI). ( A ) WT ES cells were treated with 50 μM Cr(VI) for 6 hr or 50 μM H 2 O 2 for 2 hr. The cells were labeled with 10 μM CM-H 2 DCFDA for 30 min and were subjected to flow cytometric analysis. DCF-positive cells were identified by CellQuest analysis. Cells were either pretreated with 5 mM NAC for 4 hr or left untreated prior to exposure to 50 μM Cr(VI) for various times. ( B ) Western blotting was used to measure the expression of HO-1 and actin in WT ES cells treated with Cr(VI) 50 μM for various times. ( C ) The relative levels of HO-1 protein expression were quantified by gel imaging in ES cells with or without NAC and Cr(VI) treatment. ( D ) Cells exposed to Cr(VI) at 50 μM for various times were analyzed for the cellular level of GSSG. Western blot was done to measure ( E ) phosphorylated and total JNK, p38, and ERK and ( F ) p-JUN, p-ATF2, c-FOS, and actin. The results were quantified by chemiluminescence imaging and the fold induction of p-JNK over JNK, p-38 over P38, p-ERK over ERK, and p-c-Jun, p-ATF2, and c-Fos over actin was calculated. ( G ) WT, Map2k4 (−/−) , and Map2k7 (−/−) ES cells were either pretreated with 5 mM NAC for 4 hr or left untreated prior to exposure to 50 μM Cr(VI) for 8 hr. Cell survival was measured by the MTS assay. ( H ) WT cells were exposed to 1 μM or 10 μM Cr(VI) in the presence or absence of 5 mM NAC. Colonies were counted on day 8 of treatment. The values in B, D, and E are shown as mean ± SD from at least three experiments. The ratio of phosphoprotein versus total protein or actin was calculated in each sample, and the fold induction was calculated in comparison with the untreated control of the same cell type. * p
Figure Legend Snippet: NAC suppresses MAPK activation and cytotoxicity induced by high-concentration Cr(VI). ( A ) WT ES cells were treated with 50 μM Cr(VI) for 6 hr or 50 μM H 2 O 2 for 2 hr. The cells were labeled with 10 μM CM-H 2 DCFDA for 30 min and were subjected to flow cytometric analysis. DCF-positive cells were identified by CellQuest analysis. Cells were either pretreated with 5 mM NAC for 4 hr or left untreated prior to exposure to 50 μM Cr(VI) for various times. ( B ) Western blotting was used to measure the expression of HO-1 and actin in WT ES cells treated with Cr(VI) 50 μM for various times. ( C ) The relative levels of HO-1 protein expression were quantified by gel imaging in ES cells with or without NAC and Cr(VI) treatment. ( D ) Cells exposed to Cr(VI) at 50 μM for various times were analyzed for the cellular level of GSSG. Western blot was done to measure ( E ) phosphorylated and total JNK, p38, and ERK and ( F ) p-JUN, p-ATF2, c-FOS, and actin. The results were quantified by chemiluminescence imaging and the fold induction of p-JNK over JNK, p-38 over P38, p-ERK over ERK, and p-c-Jun, p-ATF2, and c-Fos over actin was calculated. ( G ) WT, Map2k4 (−/−) , and Map2k7 (−/−) ES cells were either pretreated with 5 mM NAC for 4 hr or left untreated prior to exposure to 50 μM Cr(VI) for 8 hr. Cell survival was measured by the MTS assay. ( H ) WT cells were exposed to 1 μM or 10 μM Cr(VI) in the presence or absence of 5 mM NAC. Colonies were counted on day 8 of treatment. The values in B, D, and E are shown as mean ± SD from at least three experiments. The ratio of phosphoprotein versus total protein or actin was calculated in each sample, and the fold induction was calculated in comparison with the untreated control of the same cell type. * p

Techniques Used: Activation Assay, Concentration Assay, Labeling, Flow Cytometry, Western Blot, Expressing, Imaging, MTS Assay

16) Product Images from "Anthrax Lethal Toxin Impairs IL-8 Expression in Epithelial Cells through Inhibition of Histone H3 Modification"

Article Title: Anthrax Lethal Toxin Impairs IL-8 Expression in Epithelial Cells through Inhibition of Histone H3 Modification

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000359

LT inhibits IL-8 expression via a MAPK-dependent pathway in Beas-2B cells. (A) Beas-2B cells were incubated for 1 h with LT (1 µg/ml) and stimulated with TNFα (10 ng/ml) for an additional 2 h. Western blot analyses were performed using antibodies directed against ERK, phospho-ERK (ERKpT202-Y204), p38, phospho-p38 (p38pT180-Y182), JNK, and phospho-JNK (JNKpT183-Y185) and were normalized with ß-actin antibody. (B) Cells were pretreated with SB203580 (10 µM) or PD98059 (10 µM) for 1 h before incubation with TNFα (10 ng/ml). IL-8 concentrations were measured in supernatants after 24 h stimulation. (C) Cells were incubated for 1 h with LT (1 µg/ml) and stimulated by TNFα (10 ng/ml) for an additional 2 h. Western blot analyses were performed using antibodies directed against MEK-1, MEK-2, and MEK-3 and were normalized with ß-actin antibody. (D) Cells were transfected with an IL-8 promoter reporter plasmid, and then incubated for 1 h with LT (1 µg/ml), SB203580 (10 µM), or PD98059 (10 µM) before addition of TNFα (10 ng/ml). Luciferase activity was then measured after 24 h stimulation. (E) Cells were incubated for 1 h with LT (1 µg/ml) and stimulated by TNFα (10 ng/ml) for an additional 45 min. Western blot analyses were performed using antibodies directed against MSK-2 and phospho-MSK-2 and normalized with ß-actin antibody.
Figure Legend Snippet: LT inhibits IL-8 expression via a MAPK-dependent pathway in Beas-2B cells. (A) Beas-2B cells were incubated for 1 h with LT (1 µg/ml) and stimulated with TNFα (10 ng/ml) for an additional 2 h. Western blot analyses were performed using antibodies directed against ERK, phospho-ERK (ERKpT202-Y204), p38, phospho-p38 (p38pT180-Y182), JNK, and phospho-JNK (JNKpT183-Y185) and were normalized with ß-actin antibody. (B) Cells were pretreated with SB203580 (10 µM) or PD98059 (10 µM) for 1 h before incubation with TNFα (10 ng/ml). IL-8 concentrations were measured in supernatants after 24 h stimulation. (C) Cells were incubated for 1 h with LT (1 µg/ml) and stimulated by TNFα (10 ng/ml) for an additional 2 h. Western blot analyses were performed using antibodies directed against MEK-1, MEK-2, and MEK-3 and were normalized with ß-actin antibody. (D) Cells were transfected with an IL-8 promoter reporter plasmid, and then incubated for 1 h with LT (1 µg/ml), SB203580 (10 µM), or PD98059 (10 µM) before addition of TNFα (10 ng/ml). Luciferase activity was then measured after 24 h stimulation. (E) Cells were incubated for 1 h with LT (1 µg/ml) and stimulated by TNFα (10 ng/ml) for an additional 45 min. Western blot analyses were performed using antibodies directed against MSK-2 and phospho-MSK-2 and normalized with ß-actin antibody.

Techniques Used: Expressing, Incubation, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay

17) Product Images from "The protective effect of Prunella vulgaris ethanol extract against vascular inflammation in TNF-α-stimulated human aortic smooth muscle cells"

Article Title: The protective effect of Prunella vulgaris ethanol extract against vascular inflammation in TNF-α-stimulated human aortic smooth muscle cells

Journal: BMB Reports

doi: 10.5483/BMBRep.2013.46.7.214

P. vulgaris extract selectively inhibits production of NO and ROS activation of p38 and ERK MAPKs in TNF-α-stimulated HASMCs. HASMCs were pretreated with P. vulgaris extract (10, 50, and 250 μg/ml) for 2 h and stimulated with TNF-α (10 ng/ml) for 8 h (A) NO levels were measured as described in the Materials and Methods. (B) The level of ROS was measured as described with a microplate fluorescence reader (C) Expression and phosphorylation of ERK, p38, and JNK MAPKs were assessed by western blotting. (D) Densitometric analysis of western blots showing the relative amounts of phosphorylated and total ERK, p38, and JNK. Results are expressed as mean ± SEM (n=3). * P < 0.05, ** P < 0.01, *** P < 0.005 vs. control group (Con); # P < 0.05, ## P < 0.01, ### P < 0.005 vs. TNF-α-only treatment group.
Figure Legend Snippet: P. vulgaris extract selectively inhibits production of NO and ROS activation of p38 and ERK MAPKs in TNF-α-stimulated HASMCs. HASMCs were pretreated with P. vulgaris extract (10, 50, and 250 μg/ml) for 2 h and stimulated with TNF-α (10 ng/ml) for 8 h (A) NO levels were measured as described in the Materials and Methods. (B) The level of ROS was measured as described with a microplate fluorescence reader (C) Expression and phosphorylation of ERK, p38, and JNK MAPKs were assessed by western blotting. (D) Densitometric analysis of western blots showing the relative amounts of phosphorylated and total ERK, p38, and JNK. Results are expressed as mean ± SEM (n=3). * P < 0.05, ** P < 0.01, *** P < 0.005 vs. control group (Con); # P < 0.05, ## P < 0.01, ### P < 0.005 vs. TNF-α-only treatment group.

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

18) Product Images from "Low disabled-2 expression promotes tumor progression and determines poor survival and high recurrence of esophageal squamous cell carcinoma"

Article Title: Low disabled-2 expression promotes tumor progression and determines poor survival and high recurrence of esophageal squamous cell carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.8460

The low-DAB2 esophageal cancer cells had a higher phosphorylated ERK (p-ERK) expression and migration abilities A. The western blot analysis of DAB2 protein in five ESCC cell lines (KYSE) and human normal esophageal squamous epithelial cell line (Het-1A). B. The wound healing assay (upper panel) and transwell migration assay (lower panel) to evaluate the horizontal and vertical migration abilities in ESCC cell lines. C. TheERK, p-ERK and β-catenin levels in ESCC cell lines. D. The quantitative representation of migration abilities in ESCC cell lines.
Figure Legend Snippet: The low-DAB2 esophageal cancer cells had a higher phosphorylated ERK (p-ERK) expression and migration abilities A. The western blot analysis of DAB2 protein in five ESCC cell lines (KYSE) and human normal esophageal squamous epithelial cell line (Het-1A). B. The wound healing assay (upper panel) and transwell migration assay (lower panel) to evaluate the horizontal and vertical migration abilities in ESCC cell lines. C. TheERK, p-ERK and β-catenin levels in ESCC cell lines. D. The quantitative representation of migration abilities in ESCC cell lines.

Techniques Used: Expressing, Migration, Western Blot, Wound Healing Assay, Transwell Migration Assay

19) Product Images from "HDAC8 regulates a stress response pathway in melanoma to mediate escape from BRAF inhibitor therapy"

Article Title: HDAC8 regulates a stress response pathway in melanoma to mediate escape from BRAF inhibitor therapy

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-19-0040

HDAC8 modulates BRAF inhibitor sensitivity through regulation of RTK/RAS/RAF/MEK/ERK signaling A) Introduction of HDAC8 increases basal phospho-ERK levels in melanoma cells and MAPK signaling under BRAF inhibitor therapy. Control (EV) and HDAC8 expressing melanoma cells 1205Lu (3 μM) and WM164 (1 μM) were treated with vemurafenib for increasing periods of time (0–48 hr) before being subjected to Western Blotting for phospho-ERK (p-ERK) and total ERK expression. B) Quantification of phospho-ERK levels relative to total ERK levels. C) HDAC8 introduction is associated with increased CRAF phosphorylation following BRAF inhibition. WM164 cells were treated with vemurafenib (1 μM) for increasing periods of time (0–48 hours) and probed for phospho-CRAF (S338, p-CRAF) and CRAF expression. D) HDAC8 introduction increases Ras signaling. Isogenic (EV and HDAC8 expressing) WM164 (1 μM) and 1205Lu (3 μM) cells were treated for vemurafenib for 24 hours and probed for activated Ras (GTP-bound) and total Ras. E-F) Introduction of HDAC8 increases baseline RTK signaling. Isogenic pairs of E) 1205Lu and F) WM164 cells were analyzed using a phospho-RTK array. Resulting membranes were scanned and densitometry was performed (ND symbolizes none detected). Data show the increase in phosphorylation of the RTKs EGFR(p-EGFR), c-MET(p-c-MET), and FGFR3 (p-FGFR3) following HDAC8 introduction. G) EGFR inhibition restores BRAF inhibitor sensitivity in cell lines that express HDAC8. Isogenic 1205Lu and WM164 melanoma cells were treated with vehicle, vemurafenib (BRAFi, 3 μM for 1205Lu, 1 μM for WM164), erolitinib (EGFRi, 1 μM) or both drugs in combination (BRAFi/EGFRi) for 72 hours and apoptosis was measured by Annexin V staining and flow cytometry. Significance was determined with a one way ANOVA followed by a post hoc t test with #=p > 0.05 (non significant), **=p
Figure Legend Snippet: HDAC8 modulates BRAF inhibitor sensitivity through regulation of RTK/RAS/RAF/MEK/ERK signaling A) Introduction of HDAC8 increases basal phospho-ERK levels in melanoma cells and MAPK signaling under BRAF inhibitor therapy. Control (EV) and HDAC8 expressing melanoma cells 1205Lu (3 μM) and WM164 (1 μM) were treated with vemurafenib for increasing periods of time (0–48 hr) before being subjected to Western Blotting for phospho-ERK (p-ERK) and total ERK expression. B) Quantification of phospho-ERK levels relative to total ERK levels. C) HDAC8 introduction is associated with increased CRAF phosphorylation following BRAF inhibition. WM164 cells were treated with vemurafenib (1 μM) for increasing periods of time (0–48 hours) and probed for phospho-CRAF (S338, p-CRAF) and CRAF expression. D) HDAC8 introduction increases Ras signaling. Isogenic (EV and HDAC8 expressing) WM164 (1 μM) and 1205Lu (3 μM) cells were treated for vemurafenib for 24 hours and probed for activated Ras (GTP-bound) and total Ras. E-F) Introduction of HDAC8 increases baseline RTK signaling. Isogenic pairs of E) 1205Lu and F) WM164 cells were analyzed using a phospho-RTK array. Resulting membranes were scanned and densitometry was performed (ND symbolizes none detected). Data show the increase in phosphorylation of the RTKs EGFR(p-EGFR), c-MET(p-c-MET), and FGFR3 (p-FGFR3) following HDAC8 introduction. G) EGFR inhibition restores BRAF inhibitor sensitivity in cell lines that express HDAC8. Isogenic 1205Lu and WM164 melanoma cells were treated with vehicle, vemurafenib (BRAFi, 3 μM for 1205Lu, 1 μM for WM164), erolitinib (EGFRi, 1 μM) or both drugs in combination (BRAFi/EGFRi) for 72 hours and apoptosis was measured by Annexin V staining and flow cytometry. Significance was determined with a one way ANOVA followed by a post hoc t test with #=p > 0.05 (non significant), **=p

Techniques Used: Expressing, Western Blot, Inhibition, Staining, Flow Cytometry

HDAC8 deacetylates the transcription factor c-Jun at lysine 273 leading to increased transcriptional activity, increased phospho-ERK signaling and BRAF inhibitor resistance. A) HDAC8 introduction is associated with a unique gene signature. Heatmap of an RNA-seq analysis of WM164 and 1205Lu melanoma cells introduced with either empty vector (EV) or HDAC8. B) GSEA analysis identifies an AP-1 gene signature is upregulated in melanoma cells expressing HDAC8. C) HDAC8 expression increases phospho-c-Jun levels following BRAF inhibition. Isogenic WM164 cells were treated with vemurafenib (1 μM, 0–48 hours) and probed for phospho-c-Jun (p-c-Jun) and c-JUN by Western Blot. D) HDAC8 expression leads to increased c-Jun transcriptional activation following BRAF inhibition. Isogenic 1205Lu cells were transiently transfected with a c-Jun- (TRE) or ATF2- (JUN2) targeted promoter luciferase constructs and treated for 4 hours with vehicle (VC) or vemurafenib (3μM, BRAFi). Luciferase levels were measured and quantified. E) HDAC8 decreases c-Jun acetylation. Total c-JUN was immunoprecipitated (ip) from isogenic 1205Lu (EV) and 1205Lu-HDAC8 cells and blotted for total protein acetylation (ac-c-Jun). c-Jun was used as an input control. F) Structure of c-Jun identifying 3 potential acetylation sites (at lysine residues) in the DNA binding domain. G) Acetylation-deficient c-Jun at residue 273 increases melanoma cell survival following BRAF inhibition. 1205Lu cells expressing acetyl mutants of c-Jun (K268R, K271R, or K273R) in addition to WT c-Jun were treated with vemurafenib (3 μM: 72 hrs) before being analyzed for annexin V positivity by flow cytometry. H) Acetylation-deficient c-Jun at residue 273 leads to increased phospho-ERK (p-ERK) with decreased levels of BIM. Isogenic 1205Lu cells expressing WT, K268R, K271R, K273R constructs of c-Jun were treated with vemurafenib (3 μM: 0–24 hr) and probed for c-Jun, HDAC8, phospho-ERK, ERK, and BIM expression by Western Blot. I) Mutating lysine 273 of c-Jun confers increased binding to DNA. Isogenic 1205Lu cells expressing WT, K268R, K271R, K273R constructs of c-Jun as well as a negative control (NC) and positive control (PC) were incubated with a plate- bound consensus DNA sequence of c-Jun and read at a wavelength of 450 nm. J) Mutating lysine 273 of c-Jun increases mRNA expression of EGFR. qRT-PCR was performed on samples using primers for EGFR. Readings were normalized to GAPDH control. Experiments were performed in triplicate and significance was determined with a one way ANOVA followed by a post hoc t test with *=p
Figure Legend Snippet: HDAC8 deacetylates the transcription factor c-Jun at lysine 273 leading to increased transcriptional activity, increased phospho-ERK signaling and BRAF inhibitor resistance. A) HDAC8 introduction is associated with a unique gene signature. Heatmap of an RNA-seq analysis of WM164 and 1205Lu melanoma cells introduced with either empty vector (EV) or HDAC8. B) GSEA analysis identifies an AP-1 gene signature is upregulated in melanoma cells expressing HDAC8. C) HDAC8 expression increases phospho-c-Jun levels following BRAF inhibition. Isogenic WM164 cells were treated with vemurafenib (1 μM, 0–48 hours) and probed for phospho-c-Jun (p-c-Jun) and c-JUN by Western Blot. D) HDAC8 expression leads to increased c-Jun transcriptional activation following BRAF inhibition. Isogenic 1205Lu cells were transiently transfected with a c-Jun- (TRE) or ATF2- (JUN2) targeted promoter luciferase constructs and treated for 4 hours with vehicle (VC) or vemurafenib (3μM, BRAFi). Luciferase levels were measured and quantified. E) HDAC8 decreases c-Jun acetylation. Total c-JUN was immunoprecipitated (ip) from isogenic 1205Lu (EV) and 1205Lu-HDAC8 cells and blotted for total protein acetylation (ac-c-Jun). c-Jun was used as an input control. F) Structure of c-Jun identifying 3 potential acetylation sites (at lysine residues) in the DNA binding domain. G) Acetylation-deficient c-Jun at residue 273 increases melanoma cell survival following BRAF inhibition. 1205Lu cells expressing acetyl mutants of c-Jun (K268R, K271R, or K273R) in addition to WT c-Jun were treated with vemurafenib (3 μM: 72 hrs) before being analyzed for annexin V positivity by flow cytometry. H) Acetylation-deficient c-Jun at residue 273 leads to increased phospho-ERK (p-ERK) with decreased levels of BIM. Isogenic 1205Lu cells expressing WT, K268R, K271R, K273R constructs of c-Jun were treated with vemurafenib (3 μM: 0–24 hr) and probed for c-Jun, HDAC8, phospho-ERK, ERK, and BIM expression by Western Blot. I) Mutating lysine 273 of c-Jun confers increased binding to DNA. Isogenic 1205Lu cells expressing WT, K268R, K271R, K273R constructs of c-Jun as well as a negative control (NC) and positive control (PC) were incubated with a plate- bound consensus DNA sequence of c-Jun and read at a wavelength of 450 nm. J) Mutating lysine 273 of c-Jun increases mRNA expression of EGFR. qRT-PCR was performed on samples using primers for EGFR. Readings were normalized to GAPDH control. Experiments were performed in triplicate and significance was determined with a one way ANOVA followed by a post hoc t test with *=p

Techniques Used: Activity Assay, RNA Sequencing Assay, Plasmid Preparation, Expressing, Inhibition, Western Blot, Activation Assay, Transfection, Luciferase, Construct, Immunoprecipitation, Binding Assay, Flow Cytometry, Negative Control, Positive Control, Incubation, Sequencing, Quantitative RT-PCR

20) Product Images from "Amplification of wild type KRAS imparts resistance to crizotinib in MET exon 14 mutant non-small cell lung cancer"

Article Title: Amplification of wild type KRAS imparts resistance to crizotinib in MET exon 14 mutant non-small cell lung cancer

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

doi: 10.1158/1078-0432.CCR-18-0876

Acquired wild-type KRAS amplification in three patients with MET exon 14 mutant NSCLC at the time of crizotinib resistance. A, Axial CT images of the upper abdomen for Case #1 showing a liver metastasis at baseline (left panel) which significantly decreased in response to crizotinib at 2 months of therapy (middle panel). Fused coronal image of the PET-CT scan obtained at 4 months of therapy (right panel) demonstrated FDG-avid, enlarging mediastinal adenopathy (arrow) and a new loculated left pleural effusion (asterisk) from which the DFCI358 cell line was derived. B, Next generation sequencing (NGS, left panels) copy number plots of the KRAS locus on chromosome 12 from two crizotinib-naïve tumors from Case #1 and the crizotinib-resistant supraclavicular lymph node (bottom left panel) with estimated 55–75 copies of KRAS. Fluorescence in situ hybridization (FISH) for KRAS (red) and centromere 12 (CEN12, green) is shown at low and high power for the crizotinib naïve and resistant samples. Immunohistochemistry (IHC) for KRAS and phosphor-ERK (pERK) are also shown. C, Plasma gene copy number for several genes are displayed for Case #1 from blood taken at the time of crizotinib resistance. D, Coronal CT images for Case #2 showing a consolidated mass in the left upper lobe of the lung at baseline (left panel), which responded to crizotinib at 2 months of therapy (middle); however, progression with regrowth of left lung tumor was noted at 14 months indicative of the development of acquired resistance (right). E, NGS copy number plots of the KRAS locus on chromosome 12 and the EGFR locus on chromosome 7 are shown for the pre- and post-crizotinib samples for Case #2. Post-crizotinib sample shows estimated 25 copies of KRAS in the upper lobe. KRAS IHC before and after crizotinib treatment are also shown. F, KRAS (red) and CEN12 (green) FISH (top left) and EGFR (red) and centromere 7 (CEN7, green) FISH (top right) are shown in the crizotinib naïve tumor. Dual KRAS/EGFR (red/green, respectively) FISH in the crizotinib resistant tumor is shown (bottom panel). G, Axial CT images of the chest for Case #3 showing bilateral lung masses and a nodule at baseline (left), which partially responded to crizotinib (middle), followed by growth of one of the nodules in the right lower lobe (right, arrow), which was biopsied at the time of acquired resistance. H, NGS copy number plots of the KRAS and EGFR loci are shown for the pre- and post-crizotinib samples for Case #3. Post-crizotinib sample shows estimated 21 copies of KRAS. KRAS IHC before and after crizotinib treatment are also shown. FISH for KRAS/CEN12 (red/green, respectively), EGFR/CEN7 (yellow/green, respectively) and dual KRAS/EGFR (red/green) are shown in the crizotinib naïve and crizotinib resistant tumor samples.
Figure Legend Snippet: Acquired wild-type KRAS amplification in three patients with MET exon 14 mutant NSCLC at the time of crizotinib resistance. A, Axial CT images of the upper abdomen for Case #1 showing a liver metastasis at baseline (left panel) which significantly decreased in response to crizotinib at 2 months of therapy (middle panel). Fused coronal image of the PET-CT scan obtained at 4 months of therapy (right panel) demonstrated FDG-avid, enlarging mediastinal adenopathy (arrow) and a new loculated left pleural effusion (asterisk) from which the DFCI358 cell line was derived. B, Next generation sequencing (NGS, left panels) copy number plots of the KRAS locus on chromosome 12 from two crizotinib-naïve tumors from Case #1 and the crizotinib-resistant supraclavicular lymph node (bottom left panel) with estimated 55–75 copies of KRAS. Fluorescence in situ hybridization (FISH) for KRAS (red) and centromere 12 (CEN12, green) is shown at low and high power for the crizotinib naïve and resistant samples. Immunohistochemistry (IHC) for KRAS and phosphor-ERK (pERK) are also shown. C, Plasma gene copy number for several genes are displayed for Case #1 from blood taken at the time of crizotinib resistance. D, Coronal CT images for Case #2 showing a consolidated mass in the left upper lobe of the lung at baseline (left panel), which responded to crizotinib at 2 months of therapy (middle); however, progression with regrowth of left lung tumor was noted at 14 months indicative of the development of acquired resistance (right). E, NGS copy number plots of the KRAS locus on chromosome 12 and the EGFR locus on chromosome 7 are shown for the pre- and post-crizotinib samples for Case #2. Post-crizotinib sample shows estimated 25 copies of KRAS in the upper lobe. KRAS IHC before and after crizotinib treatment are also shown. F, KRAS (red) and CEN12 (green) FISH (top left) and EGFR (red) and centromere 7 (CEN7, green) FISH (top right) are shown in the crizotinib naïve tumor. Dual KRAS/EGFR (red/green, respectively) FISH in the crizotinib resistant tumor is shown (bottom panel). G, Axial CT images of the chest for Case #3 showing bilateral lung masses and a nodule at baseline (left), which partially responded to crizotinib (middle), followed by growth of one of the nodules in the right lower lobe (right, arrow), which was biopsied at the time of acquired resistance. H, NGS copy number plots of the KRAS and EGFR loci are shown for the pre- and post-crizotinib samples for Case #3. Post-crizotinib sample shows estimated 21 copies of KRAS. KRAS IHC before and after crizotinib treatment are also shown. FISH for KRAS/CEN12 (red/green, respectively), EGFR/CEN7 (yellow/green, respectively) and dual KRAS/EGFR (red/green) are shown in the crizotinib naïve and crizotinib resistant tumor samples.

Techniques Used: Amplification, Mutagenesis, Positron Emission Tomography, Computed Tomography, Derivative Assay, Next-Generation Sequencing, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Immunohistochemistry

Model of resistance to MET and MET/MAPK/ERK inhibition in DFCI358. A, At baseline, mutant MET in parental DFCI358 cells activates KRAS, which in turn amplifies downstream MAPK/ERK signaling resulting in enhanced transcription and release of EGFR ligands. B, Upon MET inhibition in parental DFCI358 cells, amplified KRAS sustains MAPK/ERK signaling via EGFR-ligand stimulated EGFR and other activated RTKs. C, Upon dual MET/MEK inhibition in KRAS amplified NS Control DFCI358 cells, cell viability is sustained by the PI3K/AKT pathway activated directly by amplified KRAS. D, With KRAS downregulation and MET inhibition in KRAS KD DFCI358 cells, resulting in diminished MAPK/ERK signaling, cell viability is sustained by the PI3K/AKT pathway activated by RTKs, such as IR/IGF1R, HER2 and EGFR.
Figure Legend Snippet: Model of resistance to MET and MET/MAPK/ERK inhibition in DFCI358. A, At baseline, mutant MET in parental DFCI358 cells activates KRAS, which in turn amplifies downstream MAPK/ERK signaling resulting in enhanced transcription and release of EGFR ligands. B, Upon MET inhibition in parental DFCI358 cells, amplified KRAS sustains MAPK/ERK signaling via EGFR-ligand stimulated EGFR and other activated RTKs. C, Upon dual MET/MEK inhibition in KRAS amplified NS Control DFCI358 cells, cell viability is sustained by the PI3K/AKT pathway activated directly by amplified KRAS. D, With KRAS downregulation and MET inhibition in KRAS KD DFCI358 cells, resulting in diminished MAPK/ERK signaling, cell viability is sustained by the PI3K/AKT pathway activated by RTKs, such as IR/IGF1R, HER2 and EGFR.

Techniques Used: Inhibition, Mutagenesis, Amplification

Crizotinib/trametinib-treated NS Control DFCI358 and crizotinib-treated KRAS KD DFCI358 induce PI3K/AKT via distinct mechanisms. A, Western blot of DFCI358 transduced with non-silencing control (NS) or KRAS siRNA (KRAS KD) treated with each drug at 0.5 μM, for 6 hours. HSP90 was used as a loading control. B, Quantification of Western blot signals shown in Fig. 3A for indicated markers in crizotinib-treated KRAS KD DFCI358 cells. Ratios of crizotinib-treated KRAS KD to crizotinib-treated NS control values were calculated to determine fold-change (induction of each marker) upon KRAS knock-down, which was ploted as Log 2 for each marker. C-G, Quantification of Western blot signals shown in Fig. 3A for pHER2 (C), pIRS1 (D), pAKT (E), pShc (F) and pSTAT3 (G). The induction of each marker in response to MET/MAPK/ERK inhibition by pharmaceutical (NS Control) and/or knock-down (KRAS KD) methods relative to MET inhibition only (NS Control) was assessed by calculating the ratios of crizotinib-treated KRAS KD or crizotinib/trametinib-treated NS Control relative to crizotinib-treated NS Control values for each marker. Additional inhibition of the ERBB family (lapatinib) and/or insulin family (linsitinib) receptors was assessed for the relative involvement of these pathways in resistance to MET/MAPK/ERK inhibition. The fold change was expressed as Log 2 for each marker and treatment condition. H, Co-IP study using an anti-pan-p110 antibody or IgG control in DFCI358 treated with DMSO, crizotinib (criz), trametinib (tram), crizotinib + trametinib (criz+tram), or copanlisib (copan), each at 0.5 μM for 6 hours. Interactions of p110 with KRAS or c-Raf were assessed by immunoblotting the immunoprecipitate against KRAS or c-Raf antibodies, respectively. Immunoblot against p110 was used to assess total levels of immunoprecipitated p110. Whole cell lysates (WCL) were blotted against targets of interest. I, J, Quantification of IP Western blot signals shown in Fig. 3H, expressed as Log 2 of fold change, where fold change is the ratio of each value relative to DMSO-treated control, normalized to total immunoprecipitated p110 levels and corrected for IgG background.
Figure Legend Snippet: Crizotinib/trametinib-treated NS Control DFCI358 and crizotinib-treated KRAS KD DFCI358 induce PI3K/AKT via distinct mechanisms. A, Western blot of DFCI358 transduced with non-silencing control (NS) or KRAS siRNA (KRAS KD) treated with each drug at 0.5 μM, for 6 hours. HSP90 was used as a loading control. B, Quantification of Western blot signals shown in Fig. 3A for indicated markers in crizotinib-treated KRAS KD DFCI358 cells. Ratios of crizotinib-treated KRAS KD to crizotinib-treated NS control values were calculated to determine fold-change (induction of each marker) upon KRAS knock-down, which was ploted as Log 2 for each marker. C-G, Quantification of Western blot signals shown in Fig. 3A for pHER2 (C), pIRS1 (D), pAKT (E), pShc (F) and pSTAT3 (G). The induction of each marker in response to MET/MAPK/ERK inhibition by pharmaceutical (NS Control) and/or knock-down (KRAS KD) methods relative to MET inhibition only (NS Control) was assessed by calculating the ratios of crizotinib-treated KRAS KD or crizotinib/trametinib-treated NS Control relative to crizotinib-treated NS Control values for each marker. Additional inhibition of the ERBB family (lapatinib) and/or insulin family (linsitinib) receptors was assessed for the relative involvement of these pathways in resistance to MET/MAPK/ERK inhibition. The fold change was expressed as Log 2 for each marker and treatment condition. H, Co-IP study using an anti-pan-p110 antibody or IgG control in DFCI358 treated with DMSO, crizotinib (criz), trametinib (tram), crizotinib + trametinib (criz+tram), or copanlisib (copan), each at 0.5 μM for 6 hours. Interactions of p110 with KRAS or c-Raf were assessed by immunoblotting the immunoprecipitate against KRAS or c-Raf antibodies, respectively. Immunoblot against p110 was used to assess total levels of immunoprecipitated p110. Whole cell lysates (WCL) were blotted against targets of interest. I, J, Quantification of IP Western blot signals shown in Fig. 3H, expressed as Log 2 of fold change, where fold change is the ratio of each value relative to DMSO-treated control, normalized to total immunoprecipitated p110 levels and corrected for IgG background.

Techniques Used: Western Blot, Transduction, Marker, Inhibition, Co-Immunoprecipitation Assay, Immunoprecipitation

KRAS amplified crizotinib resistant cells exhibit enhanced KRAS/MAPK/ERK signaling. A, Real time qPCR quantification of wild type KRAS copy number and transcript in DFCI358 tumor tissue, patient derived DFCI358 cell line, and non-amplified lung cancer cell lines for comparison. B, Western blot of MET inhibition and sustained downstream signaling in DFCI358 cell line following 6-hour crizotinib treatment. C, Dose-response curves of crizotinib resistant DFCI358, H596 and crizotinib sensitive Hs746T MET ) following a 6-hour treatment with DMSO, crizotinib (criz), trametinib (tram) or the combination. The mean signal (n=2) was calculated and normalized to the mean reference signal (n=6). Error bars represent standard deviation of the mean.
Figure Legend Snippet: KRAS amplified crizotinib resistant cells exhibit enhanced KRAS/MAPK/ERK signaling. A, Real time qPCR quantification of wild type KRAS copy number and transcript in DFCI358 tumor tissue, patient derived DFCI358 cell line, and non-amplified lung cancer cell lines for comparison. B, Western blot of MET inhibition and sustained downstream signaling in DFCI358 cell line following 6-hour crizotinib treatment. C, Dose-response curves of crizotinib resistant DFCI358, H596 and crizotinib sensitive Hs746T MET ) following a 6-hour treatment with DMSO, crizotinib (criz), trametinib (tram) or the combination. The mean signal (n=2) was calculated and normalized to the mean reference signal (n=6). Error bars represent standard deviation of the mean.

Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot, Inhibition, Standard Deviation

21) Product Images from "Metastasis associated protein 1 short-form stimulates Wnt1 pathway in mammary epithelial and cancer cells"

Article Title: Metastasis associated protein 1 short-form stimulates Wnt1 pathway in mammary epithelial and cancer cells

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-0907

Effect of MTA1s on ERK and GSK-3β-pathway in mammary epithelial cells ( A ) Western blot analysis of P-ERK, ERK, P-GSK-3β and GSK-3β in HC11/pcDNA and HC11/MTA1s clones. LE-Long exposure; SE-Short exposure. ( B ) Confocal analysis of phospho-ERK (red) and Phospho-GSK-3β (red) in HC11/pcDNA and HC11/MTA1s clones. Scale bar, 10 μm. ( C ) Effect of ERK inhibitor U0126 (10 μM) on the levels of phospho-GSK-3β, GSK-3β, phospho-ERK, ERK, β-catenin and Cyclin D2 in HC11/pcDNA and HC11/MTA1s clones. LE-Long exposure; SE-Short exposure. ( D ) RT-PCR analysis of the Wnt1 target genes WISP-1 and WISP-2 following treatment with the ERK inhibitor U0126 (10 μM) for 2 h in HC11/pcDNA and HC11/MTA1s clones. ( E ) Transcription status of Top-flash in HC11/MTA1s clones following treatment with the ERK inhibitor U0126 (10 μM) for 2 h. ( F ) Western blot analysis of β-catenin expression in HC11/MTA1s cells following transfection with either ERK-siRNA or GSK-3β–siRNA for 48h. ( G ) Transcription status of Top-flash in HC11/MTA1s clones following treatment with the ERK -siRNA for 48h.
Figure Legend Snippet: Effect of MTA1s on ERK and GSK-3β-pathway in mammary epithelial cells ( A ) Western blot analysis of P-ERK, ERK, P-GSK-3β and GSK-3β in HC11/pcDNA and HC11/MTA1s clones. LE-Long exposure; SE-Short exposure. ( B ) Confocal analysis of phospho-ERK (red) and Phospho-GSK-3β (red) in HC11/pcDNA and HC11/MTA1s clones. Scale bar, 10 μm. ( C ) Effect of ERK inhibitor U0126 (10 μM) on the levels of phospho-GSK-3β, GSK-3β, phospho-ERK, ERK, β-catenin and Cyclin D2 in HC11/pcDNA and HC11/MTA1s clones. LE-Long exposure; SE-Short exposure. ( D ) RT-PCR analysis of the Wnt1 target genes WISP-1 and WISP-2 following treatment with the ERK inhibitor U0126 (10 μM) for 2 h in HC11/pcDNA and HC11/MTA1s clones. ( E ) Transcription status of Top-flash in HC11/MTA1s clones following treatment with the ERK inhibitor U0126 (10 μM) for 2 h. ( F ) Western blot analysis of β-catenin expression in HC11/MTA1s cells following transfection with either ERK-siRNA or GSK-3β–siRNA for 48h. ( G ) Transcription status of Top-flash in HC11/MTA1s clones following treatment with the ERK -siRNA for 48h.

Techniques Used: Western Blot, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

MTA1s expression correlates with Wnt1, P-GSK-3β and P-ERK expression in breast cancer cell lines ( A ) Western blot analysis of MTA1s, phospho-GSK-3β, GSK-3β, P-ERK, and ERK in the human breast cancer cell lines. ( B ) Confocal analysis of Wnt1 (red), Phospho-GSK-3β (red), GSK-3β (red), P-ERK (green), ERK (green) and MTA1s in MDA-MB-435, SKBR3, T47D and ZR-75 breast cancer cell lines. Scale bar 10 μm. ( C ) Expression of Wnt1 and MTA1s in human breast tumors. Breast cancer were immunostained for Wnt1 (a–d), and MTA1s (f–i), and control serum (e,j). Four representative sets shown are derived from consecutive sections of same tumor. Higher magnification is shown as inset.
Figure Legend Snippet: MTA1s expression correlates with Wnt1, P-GSK-3β and P-ERK expression in breast cancer cell lines ( A ) Western blot analysis of MTA1s, phospho-GSK-3β, GSK-3β, P-ERK, and ERK in the human breast cancer cell lines. ( B ) Confocal analysis of Wnt1 (red), Phospho-GSK-3β (red), GSK-3β (red), P-ERK (green), ERK (green) and MTA1s in MDA-MB-435, SKBR3, T47D and ZR-75 breast cancer cell lines. Scale bar 10 μm. ( C ) Expression of Wnt1 and MTA1s in human breast tumors. Breast cancer were immunostained for Wnt1 (a–d), and MTA1s (f–i), and control serum (e,j). Four representative sets shown are derived from consecutive sections of same tumor. Higher magnification is shown as inset.

Techniques Used: Expressing, Western Blot, Multiple Displacement Amplification, Derivative Assay

22) Product Images from "Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status"

Article Title: Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status

Journal: Oncotarget

doi: 10.18632/oncotarget.19779

Phenformin enhances the anti-tumor effect of selumetinib in vitro through different mechanisms in KRAS-mutant NSCLC cell lines with alternative LKB1 status ( A ) Colony assays starting with 200 cells after incubation with DMSO (ctrl), selumetinib (S), phenformin (P) or the combination (S+P) for 2 weeks. Regardless of the LKB1 status, the combination of S and P had better growth inhibition effect than either agent alone. Please note the different ratios of S and P used for each cell line. ( B ) After 48 hours treatment, the apoptotic population was measured via flow cytometry based on 7-AAD and annexin V staining. Irrespective of LKB1 status, the combination treatment resulted in more apoptotic cells. ( C ) Histogram representation of b. ( D ) In both A549 pBabe and A549 LKB1 cells, the combination therapy potently downregulated BCL-XL level. However, only in A549 pBabe cells, the S+P combination reduced BCL-2 level more significantly than either S or P alone. ( E ) Western blot showing LKB1 inactivation resulted in lower level of p-ERK but high p-S6. Selumetinib alone potently suppressed p-ERK but upregulated p-S6 after incubation for 48 hrs. Phenformin helped suppress p-S6. Although in A549 LKB1 cells, the suppression was parallel to AMPK activation (i.e. increased p-AMPK/t-AMPK ratio), in A549 pBabe cells, no significant change in p-AMPK/t-AMPK ratio was observed.
Figure Legend Snippet: Phenformin enhances the anti-tumor effect of selumetinib in vitro through different mechanisms in KRAS-mutant NSCLC cell lines with alternative LKB1 status ( A ) Colony assays starting with 200 cells after incubation with DMSO (ctrl), selumetinib (S), phenformin (P) or the combination (S+P) for 2 weeks. Regardless of the LKB1 status, the combination of S and P had better growth inhibition effect than either agent alone. Please note the different ratios of S and P used for each cell line. ( B ) After 48 hours treatment, the apoptotic population was measured via flow cytometry based on 7-AAD and annexin V staining. Irrespective of LKB1 status, the combination treatment resulted in more apoptotic cells. ( C ) Histogram representation of b. ( D ) In both A549 pBabe and A549 LKB1 cells, the combination therapy potently downregulated BCL-XL level. However, only in A549 pBabe cells, the S+P combination reduced BCL-2 level more significantly than either S or P alone. ( E ) Western blot showing LKB1 inactivation resulted in lower level of p-ERK but high p-S6. Selumetinib alone potently suppressed p-ERK but upregulated p-S6 after incubation for 48 hrs. Phenformin helped suppress p-S6. Although in A549 LKB1 cells, the suppression was parallel to AMPK activation (i.e. increased p-AMPK/t-AMPK ratio), in A549 pBabe cells, no significant change in p-AMPK/t-AMPK ratio was observed.

Techniques Used: In Vitro, Mutagenesis, Incubation, Inhibition, Flow Cytometry, Cytometry, Staining, Western Blot, Activation Assay

23) Product Images from "SPLUNC1 Regulates Cell Progression and Apoptosis through the miR-141-PTEN/p27 Pathway, but Is Hindered by LMP1"

Article Title: SPLUNC1 Regulates Cell Progression and Apoptosis through the miR-141-PTEN/p27 Pathway, but Is Hindered by LMP1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056929

SPLUNC1 promoted cell differentiation and inhibits tumor growth. ( A ) Enforced expression of SPLUNC1 increased JNK2 and NFκB protein expression, and inhibited phosphorylation of ERK (p-Tyr-204) and Iκα (Ser-32). ΔSPLUNC1 had no effect on phosphorylation of ERK (p-Tyr-204) and Iκα (Ser-32). ( B ) SPLUNC1 also increased p27 expression and decreased the expression of CCND1, CCND2, CCND3, CCNE2, and CDK2. ( C, D ) clone formation in soft agar; tumors formed in the presence of full length SPLUNC1 or ΔSPLUNC1 were smaller versus control. ( E, F ) SPLUNC1 and ΔSPLUNC1 inhibited tumor formation in nude mice and ( G ) SPLUNC1 expression in the transplant tumor was validated by immunohistochemical analysis.
Figure Legend Snippet: SPLUNC1 promoted cell differentiation and inhibits tumor growth. ( A ) Enforced expression of SPLUNC1 increased JNK2 and NFκB protein expression, and inhibited phosphorylation of ERK (p-Tyr-204) and Iκα (Ser-32). ΔSPLUNC1 had no effect on phosphorylation of ERK (p-Tyr-204) and Iκα (Ser-32). ( B ) SPLUNC1 also increased p27 expression and decreased the expression of CCND1, CCND2, CCND3, CCNE2, and CDK2. ( C, D ) clone formation in soft agar; tumors formed in the presence of full length SPLUNC1 or ΔSPLUNC1 were smaller versus control. ( E, F ) SPLUNC1 and ΔSPLUNC1 inhibited tumor formation in nude mice and ( G ) SPLUNC1 expression in the transplant tumor was validated by immunohistochemical analysis.

Techniques Used: Cell Differentiation, Expressing, Mouse Assay, Immunohistochemistry

24) Product Images from "Impaired tumor angiogenesis and VEGF-induced pathway in endothelial CD146 knockout mice"

Article Title: Impaired tumor angiogenesis and VEGF-induced pathway in endothelial CD146 knockout mice

Journal: Protein & Cell

doi: 10.1007/s13238-014-0047-y

Inhibition of VEGF-mediated signal transduction in CD146-null ECs . (A) Phosphorylation of VEGFR-2 upon VEGF stimulation (50 ng/mL, 10 min) was determined in ECs from WT and CD146 EC-KO mice. (B) Activation of p38 induced by VEGF (50 ng/mL, 30 min) was measured in ECs from WT and CD146 EC-KO mice. (C) Degradation of I-κB and activation of NF-κB p65 induced by VEGF (50 ng/mL, 7 h) were determined in ECs from WT and CD146 EC-KO mice. (D and E) AKT and ERK activation induced by VEGF (50 ng/mL, 30 min) were measured in ECs isolated from WT and CD146 EC-KO mice. All Western blots were quantified by measuring the band density. Bar graphs (mean ± SD) present normalized values from at least 3 independent experiments. ***, P
Figure Legend Snippet: Inhibition of VEGF-mediated signal transduction in CD146-null ECs . (A) Phosphorylation of VEGFR-2 upon VEGF stimulation (50 ng/mL, 10 min) was determined in ECs from WT and CD146 EC-KO mice. (B) Activation of p38 induced by VEGF (50 ng/mL, 30 min) was measured in ECs from WT and CD146 EC-KO mice. (C) Degradation of I-κB and activation of NF-κB p65 induced by VEGF (50 ng/mL, 7 h) were determined in ECs from WT and CD146 EC-KO mice. (D and E) AKT and ERK activation induced by VEGF (50 ng/mL, 30 min) were measured in ECs isolated from WT and CD146 EC-KO mice. All Western blots were quantified by measuring the band density. Bar graphs (mean ± SD) present normalized values from at least 3 independent experiments. ***, P

Techniques Used: Inhibition, Transduction, Mouse Assay, Activation Assay, Isolation, Western Blot

25) Product Images from "Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling"

Article Title: Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200403020

Acinus formation requires JNK activity. (A, i) Fluorescence microscopic analysis of 4 d acini cultured in the absence (−) or presence of SP600125 (50 μM) or RU486 (50 μM); staining with DAPI (top), rhodamine-phalloidin and DAPI (second from top; both 40X; bar, 10 μm), anti–E-cadherin (FITC) and rhodamine-phalloidin (third from top; 20X; bar, 20 μm), anti–β-catenin and DAPI (fourth from top), anti–β4-integrin (FITC) and rhodamine-phalloidin (second from bottom; both 40X; bar, 10 μm) and anti-ZO1 and DAPI (bottom; 20X; bar, 20 μm). (ii) 2 d acini formed in presence of PD98059 (10 μM), SB230580 (1 μM), and SP600125 (50 μM) stained with Calcein AM and EtBr. Bar, 10 μm. (B) EMSA analysis, using AP1 (left) and NFκB (right) binding elements, on extracts from acini treated with the JNK inhibitor, SP600125 (50 μM), for 1, 4, and 24 h. (C) Western analysis of phospho(thr183/tyr185)-JNK and JNK in extracts from cells at harvest (H), cells maintained as a monolayer on plastic (M), 2 and 4 d acini, and 2 d acini in the absence of hydrocortisone (2d-HC). (D) Western analysis of phospho(ser63)- c-Jun (i), c-Jun (ii), phospho(thr183/tyr185)-JNK (iii), JNK (iv), phospho(thr202/tyr204)-ERK (v), and ERK (vi) in 2 and 4 d acini and 2 d acini cultured in the presence of SP600125.
Figure Legend Snippet: Acinus formation requires JNK activity. (A, i) Fluorescence microscopic analysis of 4 d acini cultured in the absence (−) or presence of SP600125 (50 μM) or RU486 (50 μM); staining with DAPI (top), rhodamine-phalloidin and DAPI (second from top; both 40X; bar, 10 μm), anti–E-cadherin (FITC) and rhodamine-phalloidin (third from top; 20X; bar, 20 μm), anti–β-catenin and DAPI (fourth from top), anti–β4-integrin (FITC) and rhodamine-phalloidin (second from bottom; both 40X; bar, 10 μm) and anti-ZO1 and DAPI (bottom; 20X; bar, 20 μm). (ii) 2 d acini formed in presence of PD98059 (10 μM), SB230580 (1 μM), and SP600125 (50 μM) stained with Calcein AM and EtBr. Bar, 10 μm. (B) EMSA analysis, using AP1 (left) and NFκB (right) binding elements, on extracts from acini treated with the JNK inhibitor, SP600125 (50 μM), for 1, 4, and 24 h. (C) Western analysis of phospho(thr183/tyr185)-JNK and JNK in extracts from cells at harvest (H), cells maintained as a monolayer on plastic (M), 2 and 4 d acini, and 2 d acini in the absence of hydrocortisone (2d-HC). (D) Western analysis of phospho(ser63)- c-Jun (i), c-Jun (ii), phospho(thr183/tyr185)-JNK (iii), JNK (iv), phospho(thr202/tyr204)-ERK (v), and ERK (vi) in 2 and 4 d acini and 2 d acini cultured in the presence of SP600125.

Techniques Used: Activity Assay, Fluorescence, Cell Culture, Staining, Binding Assay, Western Blot

26) Product Images from "Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin"

Article Title: Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin

Journal: Oncotarget

doi:

Apigenin counteracts TGF-β's effect on NIS reduction Western blots show NIS protein levels along with pERK, ERK, pAkt, Akt, and BRAF in PCCl3/Tet-On BRAF V600E cells. Cells were deprived of TSH for five days and then stimulated with TSH for 48 hours, followed by treatment with inhibitors at their optimal concentration, co-treated with or without 20 μM of Apigenin (AP), in the presence of 10 ng/ml TGF-β for 24 hours before protein extraction. 2 μg/ml doxycycline (dox) was added with TSH to induce oncogene expression. GAPDH served as a loading control. Arrowheads indicate hyperglycosylated (►) and hypoglycosylated (▻) NIS. Data are representative of two independent trials.
Figure Legend Snippet: Apigenin counteracts TGF-β's effect on NIS reduction Western blots show NIS protein levels along with pERK, ERK, pAkt, Akt, and BRAF in PCCl3/Tet-On BRAF V600E cells. Cells were deprived of TSH for five days and then stimulated with TSH for 48 hours, followed by treatment with inhibitors at their optimal concentration, co-treated with or without 20 μM of Apigenin (AP), in the presence of 10 ng/ml TGF-β for 24 hours before protein extraction. 2 μg/ml doxycycline (dox) was added with TSH to induce oncogene expression. GAPDH served as a loading control. Arrowheads indicate hyperglycosylated (►) and hypoglycosylated (▻) NIS. Data are representative of two independent trials.

Techniques Used: Western Blot, Concentration Assay, Protein Extraction, Expressing

27) Product Images from "A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis"

Article Title: A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis

Journal: Oncotarget

doi: 10.18632/oncotarget.14377

PKCA-induced phosphorylation of PKCδ, MAPK/ERK and induction of Fli-1 transcriptional activation can be reversed by the PKCδ-inhibitor Rottlerin A . A75 or TPA treatment (2μM) for 24 hours induces phosphorylation of PKCδ in both HEL and CB7 cell lines. B . Phosphorylation of MAPK/ERK and PKCδ in HEL cells treated with indicated compounds with or without PKCδ inhibitor Rottlerin. C . Fli-1 transcriptional activity induced by A75 and TPA compounds in HEK293T cells co-transfected with FB-Luc+MigR1-Fli-1 is blocked by Rottlerin. D . Extracts from CB7 and HEL cells were immunoprecipitated using anti-Fli-1 antibody followed by immune-blotting with anti-phospho-Serine/Threonine or anti-Fli-1 antibodies.
Figure Legend Snippet: PKCA-induced phosphorylation of PKCδ, MAPK/ERK and induction of Fli-1 transcriptional activation can be reversed by the PKCδ-inhibitor Rottlerin A . A75 or TPA treatment (2μM) for 24 hours induces phosphorylation of PKCδ in both HEL and CB7 cell lines. B . Phosphorylation of MAPK/ERK and PKCδ in HEL cells treated with indicated compounds with or without PKCδ inhibitor Rottlerin. C . Fli-1 transcriptional activity induced by A75 and TPA compounds in HEK293T cells co-transfected with FB-Luc+MigR1-Fli-1 is blocked by Rottlerin. D . Extracts from CB7 and HEL cells were immunoprecipitated using anti-Fli-1 antibody followed by immune-blotting with anti-phospho-Serine/Threonine or anti-Fli-1 antibodies.

Techniques Used: Activation Assay, Activity Assay, Transfection, Immunoprecipitation

28) Product Images from "Concomitant activation of ETS-like transcription factor-1 and Death Receptor-5 via extracellular signal-regulated kinase in withaferin A-mediated inhibition of hepatocarcinogenesis in mice"

Article Title: Concomitant activation of ETS-like transcription factor-1 and Death Receptor-5 via extracellular signal-regulated kinase in withaferin A-mediated inhibition of hepatocarcinogenesis in mice

Journal: Scientific Reports

doi: 10.1038/s41598-017-18190-4

WA activates RSK in an ERK-dependent manner. ( A , B ) Immunoblot analysis of phosphorylated-RSK-Ser380 (pRSK) and total RSK in Huh7 and HepG2 cells treated with 5 µM WA as indicated. * p
Figure Legend Snippet: WA activates RSK in an ERK-dependent manner. ( A , B ) Immunoblot analysis of phosphorylated-RSK-Ser380 (pRSK) and total RSK in Huh7 and HepG2 cells treated with 5 µM WA as indicated. * p

Techniques Used:

Withaferin A treatment inhibits DEN-induced liver tumor formation in C57BL/6 mice. C57BL/6 mice were given intraperitoneal injection of DEN (25 mg/kg/body weight) to develop liver tumors. Tumor bearing mice were randomized to vehicle or WA treatment groups and oral administration of vehicle or WA continued for 36 or 48 hours. ( A ) Representative pictures showing macroscopic changes in the liver from DEN-C57BL6 mice treated with vehicle or WA for 36 and 48 weeks. Arrows point to tumors in untreated group. ( B ) Representative images of H E staining of liver tumors from vehicle and WA treated groups. ( C ) Bar diagram for (i) tumor incidence, (ii) tumor multiplicity and (iii) tumor size for vehicle-treated and WA-treated DEN-C57BL6 mice (n = 8 mice/group). ( D ) Tumors were subjected to immunohistochemical analysis using pERK, pELK1, pRSK, and DR5 antibodies. Columns, mean (n = 8). ( E ) Tumor lysates were immunoblotted for Bax, cleaved Caspase 3, cleaved PARP, PCNA and Ki67 as indicated. ( F ) Tumor lysates were subjected to immunoblot analyses using phosphorylated and total ERK, ELK1, RSK and DR5 as indicated. β actin was used as loading control.
Figure Legend Snippet: Withaferin A treatment inhibits DEN-induced liver tumor formation in C57BL/6 mice. C57BL/6 mice were given intraperitoneal injection of DEN (25 mg/kg/body weight) to develop liver tumors. Tumor bearing mice were randomized to vehicle or WA treatment groups and oral administration of vehicle or WA continued for 36 or 48 hours. ( A ) Representative pictures showing macroscopic changes in the liver from DEN-C57BL6 mice treated with vehicle or WA for 36 and 48 weeks. Arrows point to tumors in untreated group. ( B ) Representative images of H E staining of liver tumors from vehicle and WA treated groups. ( C ) Bar diagram for (i) tumor incidence, (ii) tumor multiplicity and (iii) tumor size for vehicle-treated and WA-treated DEN-C57BL6 mice (n = 8 mice/group). ( D ) Tumors were subjected to immunohistochemical analysis using pERK, pELK1, pRSK, and DR5 antibodies. Columns, mean (n = 8). ( E ) Tumor lysates were immunoblotted for Bax, cleaved Caspase 3, cleaved PARP, PCNA and Ki67 as indicated. ( F ) Tumor lysates were subjected to immunoblot analyses using phosphorylated and total ERK, ELK1, RSK and DR5 as indicated. β actin was used as loading control.

Techniques Used: Mouse Assay, Injection, Staining, Immunohistochemistry

ERK is important for WA-mediated ELK1 activation in HCC cells. ( A , B ) Immunoblot analysis of phosphorylated-ELK1and total ELK1 in Huh7 and HepG2 cells treated with 5 µM WA as indicated. ( C ) Nuclear and cytoplasmic lysates from WA-treated HCC cells were immunoblotted for phosphorylated-ELK1and total ELK1. * p
Figure Legend Snippet: ERK is important for WA-mediated ELK1 activation in HCC cells. ( A , B ) Immunoblot analysis of phosphorylated-ELK1and total ELK1 in Huh7 and HepG2 cells treated with 5 µM WA as indicated. ( C ) Nuclear and cytoplasmic lysates from WA-treated HCC cells were immunoblotted for phosphorylated-ELK1and total ELK1. * p

Techniques Used: Activation Assay

WA treatment increases DR5 expression in HCC cells in an ERK-dependent manner and DR5 and Ki67 exhibit inverse correlation. ( A ) Huh7 and HepG2 cells were treated with 5 µM WA and analyzed for DR5 expression in immunoblot analysis. * p
Figure Legend Snippet: WA treatment increases DR5 expression in HCC cells in an ERK-dependent manner and DR5 and Ki67 exhibit inverse correlation. ( A ) Huh7 and HepG2 cells were treated with 5 µM WA and analyzed for DR5 expression in immunoblot analysis. * p

Techniques Used: Expressing

29) Product Images from "NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING"

Article Title: NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING

Journal: Immunity

doi: 10.1016/j.immuni.2014.01.010

NLRC3 deficiency enhances immune signaling (A) Immunoblot of phosphorylated (p-) TBK1, IRF3, p65, JNK, ERK and p38 in lysates of WT and Nlrc3 −/− MEFs infected with HSV-1 (MOI 1) for indicated time points. Densitometric measurements are depicted to the right. (B) BMDMs isolated from WT or Nlrc3 −/− mice were infected with HSV-1 (MOI 1) for 2.5 hours. Cells were fixed and stained for endogenous p65 (red) or hoechst which stains the nucleus (blue). The merged purple color is indicative of nuclear p65. (C) Similar to (A), except cells were transfected with ISD. (D) Similar to (A) except cells were transfected with poly(I:C). Data are representative of at least two independent experiments.
Figure Legend Snippet: NLRC3 deficiency enhances immune signaling (A) Immunoblot of phosphorylated (p-) TBK1, IRF3, p65, JNK, ERK and p38 in lysates of WT and Nlrc3 −/− MEFs infected with HSV-1 (MOI 1) for indicated time points. Densitometric measurements are depicted to the right. (B) BMDMs isolated from WT or Nlrc3 −/− mice were infected with HSV-1 (MOI 1) for 2.5 hours. Cells were fixed and stained for endogenous p65 (red) or hoechst which stains the nucleus (blue). The merged purple color is indicative of nuclear p65. (C) Similar to (A), except cells were transfected with ISD. (D) Similar to (A) except cells were transfected with poly(I:C). Data are representative of at least two independent experiments.

Techniques Used: Infection, Isolation, Mouse Assay, Staining, Transfection

30) Product Images from "Urban particulate matter triggers lung inflammation via the ROS-MAPK-NF-κB signaling pathway"

Article Title: Urban particulate matter triggers lung inflammation via the ROS-MAPK-NF-κB signaling pathway

Journal: Journal of Thoracic Disease

doi: 10.21037/jtd.2017.09.135

NAC attenuates activation of PM-induced MAPK pathway. (A) HBECs were treated with 300 µg/cm 3 PM in a time-dependent (0.5, 1, 3 and 6 h) manner. The phosphorylation of ERK, JNK, and p38 MAPK was detected by western blotting. The optical densities of p-ERK/ERK, p-JNK/JNK and p-p38 MAPK/p38 MAPK bands are shown in (B). Values are the mean ± SEM; **, P
Figure Legend Snippet: NAC attenuates activation of PM-induced MAPK pathway. (A) HBECs were treated with 300 µg/cm 3 PM in a time-dependent (0.5, 1, 3 and 6 h) manner. The phosphorylation of ERK, JNK, and p38 MAPK was detected by western blotting. The optical densities of p-ERK/ERK, p-JNK/JNK and p-p38 MAPK/p38 MAPK bands are shown in (B). Values are the mean ± SEM; **, P

Techniques Used: Activation Assay, Western Blot

Mechanism of the PM-induced inflammatory response in the lungs (schematic). PM initially triggers oxidative stress to generate ROS and then activates the MAPK pathway. ERK, JNK and p38 MAPK further activate the NF-κB pathway to induce expression of the pro-inflammatory proteins IL-1β, IL-6, IL-8, MMP-9 and COX-2 and infiltration of inflammatory cells in lung tissues. NAC, N-acetylcysteine; PM, particulate matter; ROS, reactive oxygen species.
Figure Legend Snippet: Mechanism of the PM-induced inflammatory response in the lungs (schematic). PM initially triggers oxidative stress to generate ROS and then activates the MAPK pathway. ERK, JNK and p38 MAPK further activate the NF-κB pathway to induce expression of the pro-inflammatory proteins IL-1β, IL-6, IL-8, MMP-9 and COX-2 and infiltration of inflammatory cells in lung tissues. NAC, N-acetylcysteine; PM, particulate matter; ROS, reactive oxygen species.

Techniques Used: Expressing

Role of the MAPK pathway in the PM-induced inflammatory response in HBECs. (A) HBECs were pretreated with the ERK inhibitor U0126 (10 µM) for 30 min before PM stimulation. The mRNA expression of IL-1β, IL-6, IL-8, MMP-9, and COX-2 was detected by RT-PCR. (B) HBECs were pretreated with the JNK inhibitor SP600125 (10 µM) for 30 min before PM stimulation. The mRNA expression of IL-1β, IL-6, IL-8, MMP-9, and COX-2 was detected by RT-PCR. (C) HBECs were pretreated with the JNK inhibitor SB203580 (10 µM) for 30 min before PM stimulation. The mRNA expression of IL-1β, IL-6, IL-8, MMP-9, and COX-2 was detected by RT-PCR. Values are the mean ± SEM; *, P
Figure Legend Snippet: Role of the MAPK pathway in the PM-induced inflammatory response in HBECs. (A) HBECs were pretreated with the ERK inhibitor U0126 (10 µM) for 30 min before PM stimulation. The mRNA expression of IL-1β, IL-6, IL-8, MMP-9, and COX-2 was detected by RT-PCR. (B) HBECs were pretreated with the JNK inhibitor SP600125 (10 µM) for 30 min before PM stimulation. The mRNA expression of IL-1β, IL-6, IL-8, MMP-9, and COX-2 was detected by RT-PCR. (C) HBECs were pretreated with the JNK inhibitor SB203580 (10 µM) for 30 min before PM stimulation. The mRNA expression of IL-1β, IL-6, IL-8, MMP-9, and COX-2 was detected by RT-PCR. Values are the mean ± SEM; *, P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

31) Product Images from "NF-?B1 Inhibits TLR-Induced IFN-? Production in Macrophages Through TPL-2-dependent ERK Activation 1"

Article Title: NF-?B1 Inhibits TLR-Induced IFN-? Production in Macrophages Through TPL-2-dependent ERK Activation 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1001003

ERK inhibition augments LPS-induced IFN signaling. A) Immunoblotting of p-ERK and total ERK in total cell extracts of WT BMDM stimulated with LPS for 30 min ± PD184352 (PD). B) Immunoblotting of p-STAT1 (Y701), total STAT1, and total ERK in WT BMDM stimulated for 2h ± PD. C) Fold-induction of Cxcl9 and Nos2 mRNA in WT BMDM stimulated for 4h ± PD. Representative of three similar experiments.
Figure Legend Snippet: ERK inhibition augments LPS-induced IFN signaling. A) Immunoblotting of p-ERK and total ERK in total cell extracts of WT BMDM stimulated with LPS for 30 min ± PD184352 (PD). B) Immunoblotting of p-STAT1 (Y701), total STAT1, and total ERK in WT BMDM stimulated for 2h ± PD. C) Fold-induction of Cxcl9 and Nos2 mRNA in WT BMDM stimulated for 4h ± PD. Representative of three similar experiments.

Techniques Used: Inhibition

p50/p105-deficiency increases phosphorylation of STAT1 following TLR stimulation. A) Immunoblot of p-STAT1 (Y701) and total STAT1 in cell extracts from Il10 −/− Rag −/− and Nfkb1 −/− Il10 −/− Rag −/− BMDM following stimulation with LPS at indicated time points. ERK is included as a loading control. Numbers under the p-STAT1 band indicate the intensity of the p-STAT1 band relative to the intensity in the unstimulated Il10 −/− Rag −/− sample, normalized to total STAT1. B) Immunoblot of p-STAT1 (Y701) and total STAT1 in nuclear extracts from Nfkb1 −/− Il10 −/− Rag −/− BMDM 4h after stimulation with LPS in the presence or absence of IFN-β-specific depleting antibody. TFIID is included as a nuclear loading control. C) Fold induction of indicated IFN responsive genes in Nfkb1 −/− Il10 −/− Rag −/− BMDM 4h after stimulation with LPS in the presence or absence of an IFN-β blocking antibody. Columns represent mean values of 3 independent macrophages pools for each genotype. One of two experiments with similar results.
Figure Legend Snippet: p50/p105-deficiency increases phosphorylation of STAT1 following TLR stimulation. A) Immunoblot of p-STAT1 (Y701) and total STAT1 in cell extracts from Il10 −/− Rag −/− and Nfkb1 −/− Il10 −/− Rag −/− BMDM following stimulation with LPS at indicated time points. ERK is included as a loading control. Numbers under the p-STAT1 band indicate the intensity of the p-STAT1 band relative to the intensity in the unstimulated Il10 −/− Rag −/− sample, normalized to total STAT1. B) Immunoblot of p-STAT1 (Y701) and total STAT1 in nuclear extracts from Nfkb1 −/− Il10 −/− Rag −/− BMDM 4h after stimulation with LPS in the presence or absence of IFN-β-specific depleting antibody. TFIID is included as a nuclear loading control. C) Fold induction of indicated IFN responsive genes in Nfkb1 −/− Il10 −/− Rag −/− BMDM 4h after stimulation with LPS in the presence or absence of an IFN-β blocking antibody. Columns represent mean values of 3 independent macrophages pools for each genotype. One of two experiments with similar results.

Techniques Used: Blocking Assay

Increased IFN-signaling in TPL-2-deficient BMDM. A) Immunoblotting of p-STAT1 (Y701), total STAT1, p-ERK 1/2, or total ERK in WT, Nfkb 1 −/− , or Map3k8 −/− BMDM at indicated times following stimulation with LPS. B) Fold induction of Cxcl9 and Nos2 in WT or Map3k8 −/− BMDM 4 hours after stimulation with LPS. Each column represents 2 independent mice per genotype. One of two experiments with similar results.
Figure Legend Snippet: Increased IFN-signaling in TPL-2-deficient BMDM. A) Immunoblotting of p-STAT1 (Y701), total STAT1, p-ERK 1/2, or total ERK in WT, Nfkb 1 −/− , or Map3k8 −/− BMDM at indicated times following stimulation with LPS. B) Fold induction of Cxcl9 and Nos2 in WT or Map3k8 −/− BMDM 4 hours after stimulation with LPS. Each column represents 2 independent mice per genotype. One of two experiments with similar results.

Techniques Used: Mouse Assay

32) Product Images from "Escin protects against acetaminophen-induced liver injury in mice via attenuating inflammatory response and inhibiting ERK signaling pathway"

Article Title: Escin protects against acetaminophen-induced liver injury in mice via attenuating inflammatory response and inhibiting ERK signaling pathway

Journal: American Journal of Translational Research

doi:

Effects of escin on the hepatic ERK (A), JNK (B), p38 (C) expression and phosphorylation in APAP-induced liver injury. Mice were intraperitoneally administrated with APAP (300 mg/kg) or an equal volume of saline (control), and treated with various concentrations of escin (0, 0.5, 1, 2 and 4 mg/kg) for 30 min. Equal protein loading is illustrated by the β-actin bands. The bands were analyzed using densitometry. Each value represents mean ± SEM; n=5-6 for each group. ## P
Figure Legend Snippet: Effects of escin on the hepatic ERK (A), JNK (B), p38 (C) expression and phosphorylation in APAP-induced liver injury. Mice were intraperitoneally administrated with APAP (300 mg/kg) or an equal volume of saline (control), and treated with various concentrations of escin (0, 0.5, 1, 2 and 4 mg/kg) for 30 min. Equal protein loading is illustrated by the β-actin bands. The bands were analyzed using densitometry. Each value represents mean ± SEM; n=5-6 for each group. ## P

Techniques Used: Expressing, Mouse Assay

33) Product Images from "Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation"

Article Title: Transmodulation between Phospholipase D and c-Src Enhances Cell Proliferation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.9.3103-3115.2003

PLD enhances not only c-Src-induced tyrosine phosphorylation of paxillin but also c-Src-mediated ERK activation. COS-7 cells were transiently cotransfected for 40 h with various combinations of plasmids encoding an empty vector, PLD1, PLD2, c-Src, and c-Src plus either wild-type (wt) or catalytically inactive mutant (mt) PLD1 or PLD2. Cell lysates were immunoblotted with an antibody directed against tyrosine-phosphorylated paxillin (A) or an anti-phospho-ERK antibody (B). Expression of total paxillin and ERK was visualized by immunoblotting with antipaxillin antibody or anti-ERK antibody. Data are representative of three experiments. The intensity of phosphorylated paxillin or phospho-ERK immunoreactive bands quantified by densitometry of immunoblot was expressed as relative intensity of the bands. Results show means ± standard deviations of three independent experiments.
Figure Legend Snippet: PLD enhances not only c-Src-induced tyrosine phosphorylation of paxillin but also c-Src-mediated ERK activation. COS-7 cells were transiently cotransfected for 40 h with various combinations of plasmids encoding an empty vector, PLD1, PLD2, c-Src, and c-Src plus either wild-type (wt) or catalytically inactive mutant (mt) PLD1 or PLD2. Cell lysates were immunoblotted with an antibody directed against tyrosine-phosphorylated paxillin (A) or an anti-phospho-ERK antibody (B). Expression of total paxillin and ERK was visualized by immunoblotting with antipaxillin antibody or anti-ERK antibody. Data are representative of three experiments. The intensity of phosphorylated paxillin or phospho-ERK immunoreactive bands quantified by densitometry of immunoblot was expressed as relative intensity of the bands. Results show means ± standard deviations of three independent experiments.

Techniques Used: Activation Assay, Plasmid Preparation, Mutagenesis, Expressing

Proposed model of PLD-stimulated c-Src downstream signaling. EGF binds to its receptor, thereby stimulating both PLD and c-Src activation. It is likely that the resulting PLD activity is involved in the stimulation of Src-mediated signaling pathways such as ERK and then in the stimulation of cell proliferation. EGF induces tyrosine phosphorylation of PLD2 via c-Src and its interaction with c-Src. At present, it is not clear, however, how tyrosine phosphorylation of PLD is involved in PLD-mediated signaling mechanism.
Figure Legend Snippet: Proposed model of PLD-stimulated c-Src downstream signaling. EGF binds to its receptor, thereby stimulating both PLD and c-Src activation. It is likely that the resulting PLD activity is involved in the stimulation of Src-mediated signaling pathways such as ERK and then in the stimulation of cell proliferation. EGF induces tyrosine phosphorylation of PLD2 via c-Src and its interaction with c-Src. At present, it is not clear, however, how tyrosine phosphorylation of PLD is involved in PLD-mediated signaling mechanism.

Techniques Used: Activation Assay, Activity Assay

34) Product Images from "MIS416 Enhances Therapeutic Functions of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Against Experimental Colitis by Modulating Systemic Immune Milieu"

Article Title: MIS416 Enhances Therapeutic Functions of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Against Experimental Colitis by Modulating Systemic Immune Milieu

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01078

MIS416 does not have direct effect on the properties of human adult stem cells, including umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) including immunomodulatory functions in vitro . (A–E) hUCB-MSCs were treated with indicated concentrations of MIS416 for 24 h and further analyses were conducted. (A) Representative of bright-field microscopy images of hUCB-MSCs, bar = 500 µm. (B) Proliferation of hUCB-MSCs was determined by BrdU ELISA kit. (C) Cell cycle assay. (D) The relative expression levels of COX2, iNOS, and IDO-1 in hUCB-MSCs were analyzed by immunoblotting analysis. (E) (Left) Expression levels of RIP2, MyD88, IKK-α, and IκB-α/(Right) phosphorylation levels of NFκB p65, p38 mitogen-activated protein kinase, ERK, and JNK were determined by western blot analysis. (F) Mixed lymphocytes reaction assay of hUCB-MSCs. (G) Transwell migration assay was performed and migrated cells were quantified. Gel electrophoresis was conducted under the same experimental conditions, and images of blots were cropped. Uncropped blot images are shown in Figure S7 in Supplementary Material. Experiments were performed in triplicate. (−): negative control group, (+): ConA activated group, U: hUCB-MSCs, U + M: MIS416-treated hUCB-MSCs. **** P
Figure Legend Snippet: MIS416 does not have direct effect on the properties of human adult stem cells, including umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) including immunomodulatory functions in vitro . (A–E) hUCB-MSCs were treated with indicated concentrations of MIS416 for 24 h and further analyses were conducted. (A) Representative of bright-field microscopy images of hUCB-MSCs, bar = 500 µm. (B) Proliferation of hUCB-MSCs was determined by BrdU ELISA kit. (C) Cell cycle assay. (D) The relative expression levels of COX2, iNOS, and IDO-1 in hUCB-MSCs were analyzed by immunoblotting analysis. (E) (Left) Expression levels of RIP2, MyD88, IKK-α, and IκB-α/(Right) phosphorylation levels of NFκB p65, p38 mitogen-activated protein kinase, ERK, and JNK were determined by western blot analysis. (F) Mixed lymphocytes reaction assay of hUCB-MSCs. (G) Transwell migration assay was performed and migrated cells were quantified. Gel electrophoresis was conducted under the same experimental conditions, and images of blots were cropped. Uncropped blot images are shown in Figure S7 in Supplementary Material. Experiments were performed in triplicate. (−): negative control group, (+): ConA activated group, U: hUCB-MSCs, U + M: MIS416-treated hUCB-MSCs. **** P

Techniques Used: Derivative Assay, In Vitro, Microscopy, Enzyme-linked Immunosorbent Assay, Cell Cycle Assay, Expressing, Western Blot, Transwell Migration Assay, Nucleic Acid Electrophoresis, Negative Control

35) Product Images from "Tianfoshen oral liquid: a CFDA approved clinical traditional Chinese medicine, normalizes major cellular pathways disordered during colorectal carcinogenesis"

Article Title: Tianfoshen oral liquid: a CFDA approved clinical traditional Chinese medicine, normalizes major cellular pathways disordered during colorectal carcinogenesis

Journal: Oncotarget

doi: 10.18632/oncotarget.14675

TFS restrains hyperactive Wnt/β-catenin and MAPK signaling pathways in CRC ( A ) The activity of MAPK (ERK and JNK) signalling pathway was measured by western blotting. Quantifcation of protein level was normalized to β-actin using densitometry. ( B ) The nuclear levels of β-catenin and the protein levels of its downstream targets (c-myc and survivin) as detected by western blotting are shown (left). The quantitative analysis of WB data is shown as mean ± SD (right). ** * P
Figure Legend Snippet: TFS restrains hyperactive Wnt/β-catenin and MAPK signaling pathways in CRC ( A ) The activity of MAPK (ERK and JNK) signalling pathway was measured by western blotting. Quantifcation of protein level was normalized to β-actin using densitometry. ( B ) The nuclear levels of β-catenin and the protein levels of its downstream targets (c-myc and survivin) as detected by western blotting are shown (left). The quantitative analysis of WB data is shown as mean ± SD (right). ** * P

Techniques Used: Activity Assay, Western Blot

36) Product Images from "Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells"

Article Title: Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12077

H 2 S-stimulated endothelial NO synthase (eNOS) phosphorylation is dependent on p38 MAPK and Akt. Endothelial cells (ECs) were pre-treated with ( A ) SB203580 (10 μM), ( B ) LY294002 (10 μM), and ( C ) U0126 (10 μM) for 1 hr and then treated with NaHS (100 μM) for 30 min. Cell lysates were harvested and the level of phosphorylated forms of p38 MAPK, Akt, ERK and eNOS were measured by Western blot. n = 3, * P
Figure Legend Snippet: H 2 S-stimulated endothelial NO synthase (eNOS) phosphorylation is dependent on p38 MAPK and Akt. Endothelial cells (ECs) were pre-treated with ( A ) SB203580 (10 μM), ( B ) LY294002 (10 μM), and ( C ) U0126 (10 μM) for 1 hr and then treated with NaHS (100 μM) for 30 min. Cell lysates were harvested and the level of phosphorylated forms of p38 MAPK, Akt, ERK and eNOS were measured by Western blot. n = 3, * P

Techniques Used: Western Blot

H 2 S-induced phosphorylation of p38 MAPK, Akt and ERK. Endothelial cells (ECs) were treated with NaHS (100 μM) for different times (0–60 min.). At the end of each time-point, cells were collected and proteins lysates were analysed by Western blot, using antibodies specific for the phosphorylated and total forms of ( A ) p38 MAPK, ( B ) Akt, and ( C ) ERK. Data were normalized to total protein level, n = 3–4, * P
Figure Legend Snippet: H 2 S-induced phosphorylation of p38 MAPK, Akt and ERK. Endothelial cells (ECs) were treated with NaHS (100 μM) for different times (0–60 min.). At the end of each time-point, cells were collected and proteins lysates were analysed by Western blot, using antibodies specific for the phosphorylated and total forms of ( A ) p38 MAPK, ( B ) Akt, and ( C ) ERK. Data were normalized to total protein level, n = 3–4, * P

Techniques Used: Western Blot

37) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.306514

Ran(K152A) enhances the transformed phenotype exhibited by SKBR3 breast cancer cells. A , A single SKBR3 cell clone stably expressing either the vector-alone, wild-type Ran, or Ran(K152A) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. The error bars represent standard deviation. Representative images of the soft agar assay are shown. B , SKBR3 cells stably expressing the vector-alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved SKBR3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, and HA antibodies. Similar results were obtained using additional clones (data not shown).
Figure Legend Snippet: Ran(K152A) enhances the transformed phenotype exhibited by SKBR3 breast cancer cells. A , A single SKBR3 cell clone stably expressing either the vector-alone, wild-type Ran, or Ran(K152A) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. The error bars represent standard deviation. Representative images of the soft agar assay are shown. B , SKBR3 cells stably expressing the vector-alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved SKBR3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, and HA antibodies. Similar results were obtained using additional clones (data not shown).

Techniques Used: Transformation Assay, Stable Transfection, Expressing, Plasmid Preparation, Standard Deviation, Soft Agar Assay, Positive Control, Western Blot, Clone Assay

SMOC-2 is important for Ran promoted cellular transformation in human mammary SKBR3 adenocarcinoma cells. A , SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs (denoted as Kd 1 , 2 , or 3 ) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged, and graphed. The error bars represent S.D. B , SKBR3 cells stably expressing the vector alone, Ran(K152A), Ran(K152A), and SMOC-2 siRNA, or Ran(K152A), SMOC-2 siRNA, and an siRNA-insensitive form of SMOC-2 (SMOC-2 mutant) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, and the data were averaged and graphed as a percent of the control Ran(K152A). The error bars represent S.D. C and D , serum-starved SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs were lysed. As a positive control, an additional plate of serum-starved SKBR3 cells overexpressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies.
Figure Legend Snippet: SMOC-2 is important for Ran promoted cellular transformation in human mammary SKBR3 adenocarcinoma cells. A , SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs (denoted as Kd 1 , 2 , or 3 ) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged, and graphed. The error bars represent S.D. B , SKBR3 cells stably expressing the vector alone, Ran(K152A), Ran(K152A), and SMOC-2 siRNA, or Ran(K152A), SMOC-2 siRNA, and an siRNA-insensitive form of SMOC-2 (SMOC-2 mutant) were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, and the data were averaged and graphed as a percent of the control Ran(K152A). The error bars represent S.D. C and D , serum-starved SKBR3 cells stably expressing the vector alone, Ran(K152A), or Ran(K152A) and SMOC-2 siRNAs were lysed. As a positive control, an additional plate of serum-starved SKBR3 cells overexpressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were analyzed by Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies.

Techniques Used: Transformation Assay, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Positive Control, Western Blot

Activated Ran mutants induce cell transformation. A , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, the activated Ran mutants, or the constitutively active Ras(G12V) mutant were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. Representative images of the soft agar assay are shown. B , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved NIH-3T3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were then subjected to Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies. C , NIH-3T3 cells expressing the vector-alone or stably expressing Ran(K152A) were subjected to cell invasion assays under serum-starved and serum-stimulated conditions. The cells that invaded for each condition were counted and graphed. The results shown represent the average of three separate experiments. The error bars in A and C represent S.D.
Figure Legend Snippet: Activated Ran mutants induce cell transformation. A , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, the activated Ran mutants, or the constitutively active Ras(G12V) mutant were subjected to anchorage-independent growth (soft agar) assays. The experiments were performed three times, and the resulting colonies that formed in each experiment were counted, averaged together, and graphed. Representative images of the soft agar assay are shown. B , NIH-3T3 cells transiently expressing the vector alone, wild-type Ran, or Ran(K152A) were serum-starved overnight and lysed. As a positive control, an additional plate of serum-starved NIH-3T3 cells expressing the vector alone was stimulated with medium containing serum for 10 min prior to being lysed. The cell extracts were then subjected to Western blot analysis using phospho-ERK, phospho-JNK, HA, and vinculin antibodies. C , NIH-3T3 cells expressing the vector-alone or stably expressing Ran(K152A) were subjected to cell invasion assays under serum-starved and serum-stimulated conditions. The cells that invaded for each condition were counted and graphed. The results shown represent the average of three separate experiments. The error bars in A and C represent S.D.

Techniques Used: Transformation Assay, Expressing, Plasmid Preparation, Mutagenesis, Soft Agar Assay, Positive Control, Western Blot, Stable Transfection

38) Product Images from "Serum amyloid A inhibits RANKL-induced osteoclast formation"

Article Title: Serum amyloid A inhibits RANKL-induced osteoclast formation

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2015.83

SAA stimulates c-fms shedding by TACE, which is dependent on ERK and p38 MAPK activity. ( a ) Mouse BMDMs were pre-incubated with TAPI-1 (20 μ M ) for 1 h prior to the SAA (1 μ M ) treatment. The c-fms levels on the cell surface were determined by flow cytometry using an anti-c-fms antibody. ( b ) Mouse BMDMs were stimulated with SAA (1 μ M ) in the presence of M-CSF (30 ng ml −1 ), RANKL (100 ng ml −1 ) or M-CSF (30 ng ml −1 ) and RANKL (100 ng ml −1 ) for 0, 2, 5, 10, 30 and 60 min. The phosphorylated ERK and p38 MAPK levels were determined by immunoblotting using anti-phospho-ERK and anti-phospho-p38 MAPK antibodies. ( c ) Mouse BMDMs were pre-incubated for 1 h with U0126 (40 μ M ; top), SB203580 (20 μ M ; middle), or U0126 (40 μ M ) and SB203580 (20 μ M ; bottom) prior to the SAA (1 μ M ) treatment. The c-fms levels on the cell surface were determined by flow cytometry using an anti-c-fms antibody. The results shown are representative of three independent experiments ( a – c ). BMDM, bone marrow-derived macrophage; M-CSF, macrophage colony-stimulating factor; NT, not treated; RANKL, receptor activator of nuclear factor κB ligand; SAA, serum amyloid A.
Figure Legend Snippet: SAA stimulates c-fms shedding by TACE, which is dependent on ERK and p38 MAPK activity. ( a ) Mouse BMDMs were pre-incubated with TAPI-1 (20 μ M ) for 1 h prior to the SAA (1 μ M ) treatment. The c-fms levels on the cell surface were determined by flow cytometry using an anti-c-fms antibody. ( b ) Mouse BMDMs were stimulated with SAA (1 μ M ) in the presence of M-CSF (30 ng ml −1 ), RANKL (100 ng ml −1 ) or M-CSF (30 ng ml −1 ) and RANKL (100 ng ml −1 ) for 0, 2, 5, 10, 30 and 60 min. The phosphorylated ERK and p38 MAPK levels were determined by immunoblotting using anti-phospho-ERK and anti-phospho-p38 MAPK antibodies. ( c ) Mouse BMDMs were pre-incubated for 1 h with U0126 (40 μ M ; top), SB203580 (20 μ M ; middle), or U0126 (40 μ M ) and SB203580 (20 μ M ; bottom) prior to the SAA (1 μ M ) treatment. The c-fms levels on the cell surface were determined by flow cytometry using an anti-c-fms antibody. The results shown are representative of three independent experiments ( a – c ). BMDM, bone marrow-derived macrophage; M-CSF, macrophage colony-stimulating factor; NT, not treated; RANKL, receptor activator of nuclear factor κB ligand; SAA, serum amyloid A.

Techniques Used: Activity Assay, Incubation, Flow Cytometry, Cytometry, Derivative Assay

39) Product Images from "Combined Effects of Baicalein and Docetaxel on Apoptosis in 8505c Anaplastic Thyroid Cancer Cells via Downregulation of the ERK and Akt/mTOR Pathways"

Article Title: Combined Effects of Baicalein and Docetaxel on Apoptosis in 8505c Anaplastic Thyroid Cancer Cells via Downregulation of the ERK and Akt/mTOR Pathways

Journal: Endocrinology and Metabolism

doi: 10.3803/EnM.2018.33.1.121

Effects of the combined treatment with baicalein and docetaxel on the signaling pathway in 8505c cells. The cells were treated with baicalein and docetaxel individually or in combination for 24 hours. The expression of mammalian target of rapamycin (mTOR) (A) and the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt (B) were determined by Western blotting analysis. (C, D, E) Each histogram was presented as the mean±SEM of the band density for three independent experiments compared with β-actin expression (for mTOR) or the total expression (for ERK or Akt). a P
Figure Legend Snippet: Effects of the combined treatment with baicalein and docetaxel on the signaling pathway in 8505c cells. The cells were treated with baicalein and docetaxel individually or in combination for 24 hours. The expression of mammalian target of rapamycin (mTOR) (A) and the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt (B) were determined by Western blotting analysis. (C, D, E) Each histogram was presented as the mean±SEM of the band density for three independent experiments compared with β-actin expression (for mTOR) or the total expression (for ERK or Akt). a P

Techniques Used: Expressing, Western Blot

40) Product Images from "Dual Effects of Metformin on Adipogenic Differentiation of 3T3-L1 Preadipocyte in AMPK-Dependent and Independent Manners"

Article Title: Dual Effects of Metformin on Adipogenic Differentiation of 3T3-L1 Preadipocyte in AMPK-Dependent and Independent Manners

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061547

Dose response of 1.25–10 mM of metformin on phosphorylation of AMPK, Akt, and MAPKs in 3T3-L1 preadipocytes. Cells were cultured in differentiation medium containing 0, 1.25, 2.5, 5, or 10 mM of metformin for 15 min to examine AMPK, p38, JNK, and Akt activation and for 30 min to examine ERK activation. Results were quantified using densitometry analysis and normalized to total MAPKs, Akt, or AMPK accordingly ( n = 3). Data represent the means ± SEM from three experiments. * p
Figure Legend Snippet: Dose response of 1.25–10 mM of metformin on phosphorylation of AMPK, Akt, and MAPKs in 3T3-L1 preadipocytes. Cells were cultured in differentiation medium containing 0, 1.25, 2.5, 5, or 10 mM of metformin for 15 min to examine AMPK, p38, JNK, and Akt activation and for 30 min to examine ERK activation. Results were quantified using densitometry analysis and normalized to total MAPKs, Akt, or AMPK accordingly ( n = 3). Data represent the means ± SEM from three experiments. * p

Techniques Used: Cell Culture, Activation Assay

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Incubation:

Article Title: Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells
Article Snippet: .. The sections were blocked with 10% v/v goat serum and then incubated either with primary antibody to Ki-67 (RM-9106-S, Thermo Scientific) or the antibody to phospho-Rb protein at Ser807/811 (9308, Cell Signaling Technology) for 20 h at 4°C. .. After being rinsed in Tris-buffered saline containing 0.05% v/v Tween-20, each section was sequentially labelled with a biotinylated secondary antibody (BA-1000) and Vectastain ABC kit (PK-4000) from Vector Laboratories.

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    Cell Signaling Technology Inc anti phospho erk antibodies
    Effect of for 1 on EGF-induced activation of <t>EGFR</t> in PANC1 cells. (a, b, and c) Serum-starved PANC1 cells were stimulated with EGF (100 ng/mL) in the presence of the indicted concentrations of 1 for 5 min. Whole cell lysates were prepared and Western blotting was performed to determine the expression level of p-EGFR (a), EGFR (a), p-Akt (b), Akt (b), <t>p-ERK</t> (c), and ERK (c). α -Tubulin was used as a loading control. The blots were quantified by Image J software and the levels of p-EGFR, p-Akt, and p-ERK (normalized to EGFR, Akt, and ERK, respectively) were expressed as the mean ± SD of three independent experiments. ∗ P
    Anti Phospho Erk Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 414 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and <t>ERK</t> or phospho-MET, <t>phospho-Akt</t> and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).
    Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of AKT/pAKT, <t>mTOR/pmTOR,</t> and <t>ERK/pERK</t> in sorafenib-resistant cells The expression of pAKT, AKT, pmTOR, mTOR, pERK, and ERK in PLC/PRF5, PLC/PRF5-R1 and PLC/PRF5-R2 cells wasexamined by Western blot analysis. β-actin was used as an internal control.
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    Effect of for 1 on EGF-induced activation of EGFR in PANC1 cells. (a, b, and c) Serum-starved PANC1 cells were stimulated with EGF (100 ng/mL) in the presence of the indicted concentrations of 1 for 5 min. Whole cell lysates were prepared and Western blotting was performed to determine the expression level of p-EGFR (a), EGFR (a), p-Akt (b), Akt (b), p-ERK (c), and ERK (c). α -Tubulin was used as a loading control. The blots were quantified by Image J software and the levels of p-EGFR, p-Akt, and p-ERK (normalized to EGFR, Akt, and ERK, respectively) were expressed as the mean ± SD of three independent experiments. ∗ P

    Journal: BioMed Research International

    Article Title: Degalactotigonin, a Steroidal Glycoside from Solanum nigrum, Induces Apoptosis and Cell Cycle Arrest via Inhibiting the EGFR Signaling Pathways in Pancreatic Cancer Cells

    doi: 10.1155/2018/3120972

    Figure Lengend Snippet: Effect of for 1 on EGF-induced activation of EGFR in PANC1 cells. (a, b, and c) Serum-starved PANC1 cells were stimulated with EGF (100 ng/mL) in the presence of the indicted concentrations of 1 for 5 min. Whole cell lysates were prepared and Western blotting was performed to determine the expression level of p-EGFR (a), EGFR (a), p-Akt (b), Akt (b), p-ERK (c), and ERK (c). α -Tubulin was used as a loading control. The blots were quantified by Image J software and the levels of p-EGFR, p-Akt, and p-ERK (normalized to EGFR, Akt, and ERK, respectively) were expressed as the mean ± SD of three independent experiments. ∗ P

    Article Snippet: Anti-phospho-EGFR, anti-Akt, anti-phospho Akt (Ser473), anti-ERK, and anti-phospho-ERK antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Western Blot, Expressing, Software

    Enhanced expression of TLRs, MyD88, and MAPK pathway-associated signaling components in imDCs in response to CTX. (A–D) RT-qPCR analysis of TLR2, TLR4, TLR9, and MyD88 mRNA expression in imDCs. (E–H) Western blotting analysis of p38, p-p38, JNK, p-JNK, ERK, and p-ERK protein expression in imDCs. Error bars indicate the SEM. * P

    Journal: Frontiers in Pharmacology

    Article Title: High-Dose Cyclophosphamide Administration Orchestrates Phenotypic and Functional Alterations of Immature Dendritic Cells and Regulates Th Cell Polarization

    doi: 10.3389/fphar.2020.00775

    Figure Lengend Snippet: Enhanced expression of TLRs, MyD88, and MAPK pathway-associated signaling components in imDCs in response to CTX. (A–D) RT-qPCR analysis of TLR2, TLR4, TLR9, and MyD88 mRNA expression in imDCs. (E–H) Western blotting analysis of p38, p-p38, JNK, p-JNK, ERK, and p-ERK protein expression in imDCs. Error bars indicate the SEM. * P

    Article Snippet: Membranes were blocked with 5% milk for 1 h and incubated overnight at 4°C with primary anti-ERK, anti-phospho-ERK, anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38, and anti-β-Actin antibodies (dilution 1:1,000, Cell Signaling Technology, Boston, MA, USA), followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Cell Signaling Technology) for 30 min at 37°C.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).

    Journal: Nature Communications

    Article Title: A cysteine selenosulfide redox switch for protein chemical synthesis

    doi: 10.1038/s41467-020-16359-6

    Figure Lengend Snippet: Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).

    Article Snippet: Cell lysates were then analyzed by western blot using specific total MET (#37-0100 Invitrogen), total ERK2 (#SC-154 Tebu-bio), phospho-MET (Y1234/1235, clone CD26, #3077 Cell Signaling), phospho-Akt (S473, clone CD9E, #4060 Cell Signaling), phospho-ERK (T202/Y204, clone E10, #9106 Cell Signaling).

    Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Amplified Luminescent Proximity Homogenous Assay, Recombinant, Incubation, Standard Deviation, Phosphorylation Assay, Western Blot, Stripping Membranes, Scattering Assay, Isolation, Staining

    Expression of AKT/pAKT, mTOR/pmTOR, and ERK/pERK in sorafenib-resistant cells The expression of pAKT, AKT, pmTOR, mTOR, pERK, and ERK in PLC/PRF5, PLC/PRF5-R1 and PLC/PRF5-R2 cells wasexamined by Western blot analysis. β-actin was used as an internal control.

    Journal: Oncotarget

    Article Title: MRP3 as a novel resistance factor for sorafenib in hepatocellular carcinoma

    doi: 10.18632/oncotarget.6889

    Figure Lengend Snippet: Expression of AKT/pAKT, mTOR/pmTOR, and ERK/pERK in sorafenib-resistant cells The expression of pAKT, AKT, pmTOR, mTOR, pERK, and ERK in PLC/PRF5, PLC/PRF5-R1 and PLC/PRF5-R2 cells wasexamined by Western blot analysis. β-actin was used as an internal control.

    Article Snippet: Blots were blocked with 5% fat-free dry milk in Tris-buffered saline-Tween (TBS-T) buffer for 1 h and then incubated overnight with rabbit anti-phospho-Akt polyclonal antibody (Cell Signaling Technology, Tokyo, Japan), rabbit anti-Akt polyclonal antibody (Abcam, Cambridge, UK), rabbit anti-mTOR polyclonal antibody (Abcam), rabbit anti-phospho-mTOR (Abcam) monoclonal antibody, rabbit anti-ERK (Cell Signaling Technology) polyclonal antibody, mouse anti-phospho-ERK monoclonal antibody (Cell Signaling Technology), mouse anti-MDR1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal antibody, rabbit anti-MRP2 polyclonal antibody (Cell Signaling Technology), rabbit anti-MRP3 polyclonal antibody (Novus Biologicals, Littleton, CO), rabbit anti-MRP4 polyclonal antibody (Cell Signaling Technology), rabbit anti-BCRP polyclonal antibody (Sigma-Aldrich), rabbit anti-BSEP polyclonal antibody (Sigma-Aldrich), and rabbit anti-OCT1 polyclonal antibody (Abcam) as primary antibodies.

    Techniques: Expressing, Planar Chromatography, Western Blot