Structured Review

Millipore h89
cAMP activates mTORC1 signaling via Rheb. (A) cAMP activates mTORC1 signaling in a rapamycin-dependent manner. HEK293 cells were incubated with 10% fetal bovine serum for 36 h. After 24 h of serum deprivation, the cells were preincubated with or without serum-free medium containing 10 nM rapamycin for 45 min and were then treated with 10 μM forskolin for 5 min. The cells were lysed with buffer containing 0.5% CHAPS. Equal amounts of total cell lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (B) Forskolin activates mTORC1 in a dose-dependent manner. After 24 h of serum deprivation, HEK293 cells were treated with the indicated concentrations of forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (C) cAMP-mediated mTORC1 activation is dependent on Rheb. HEK293 cells were transfected with either the control or Rheb shRNA by using Lipofectamine. After 24 h, cells were depleted of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were immunoprecipitated (IP) with anti-Rheb antibodies. The immunoprecipitates and lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. CTL, cytotoxic T lymphocytes. (D) The level of cAMP-mediated mTORC1 activation is decreased by a Rheb dominant negative mutant. HEK293 cells were transfected with either a GFP vector or GFP-Rheb D60I by using Lipofectamine. After 24 h, cells were depleted of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (E) cAMP-mediated mTORC1 activation is not affected in TSC1-deficient cells. TSC1 +/+ and TSC1 −/− MEFs were depleted of serum for 8 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (F) Overexpression of TSC1 and TSC2 has little effect on forskolin-dependent mTORC1 activation. HEK293 cells were transfected with control or recombinant TSC1/2. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (G) Overexpression of the AMPKα1 dominant negative form does not affect forskolin-dependent mTORC1 activation. HEK293 cells were transfected with the control or recombinant form of dominant-negative AMPKα1. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. DN, dominant negative. (H) Inhibition of MAPK does not affect forskolin-dependent mTORC1 activation. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing PD98059 for 30 min and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (I) Inhibition of PKA does not affect forskolin-dependent mTORC1 activation. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing <t>H89</t> for 45 min and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments.
H89, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/h89/product/Millipore
Average 94 stars, based on 8470 article reviews
Price from $9.99 to $1999.99
h89 - by Bioz Stars, 2020-09
94/100 stars

Images

1) Product Images from "Cyclic AMP Controls mTOR through Regulation of the Dynamic Interaction between Rheb and Phosphodiesterase 4D ▿"

Article Title: Cyclic AMP Controls mTOR through Regulation of the Dynamic Interaction between Rheb and Phosphodiesterase 4D ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00217-10

cAMP activates mTORC1 signaling via Rheb. (A) cAMP activates mTORC1 signaling in a rapamycin-dependent manner. HEK293 cells were incubated with 10% fetal bovine serum for 36 h. After 24 h of serum deprivation, the cells were preincubated with or without serum-free medium containing 10 nM rapamycin for 45 min and were then treated with 10 μM forskolin for 5 min. The cells were lysed with buffer containing 0.5% CHAPS. Equal amounts of total cell lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (B) Forskolin activates mTORC1 in a dose-dependent manner. After 24 h of serum deprivation, HEK293 cells were treated with the indicated concentrations of forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (C) cAMP-mediated mTORC1 activation is dependent on Rheb. HEK293 cells were transfected with either the control or Rheb shRNA by using Lipofectamine. After 24 h, cells were depleted of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were immunoprecipitated (IP) with anti-Rheb antibodies. The immunoprecipitates and lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. CTL, cytotoxic T lymphocytes. (D) The level of cAMP-mediated mTORC1 activation is decreased by a Rheb dominant negative mutant. HEK293 cells were transfected with either a GFP vector or GFP-Rheb D60I by using Lipofectamine. After 24 h, cells were depleted of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (E) cAMP-mediated mTORC1 activation is not affected in TSC1-deficient cells. TSC1 +/+ and TSC1 −/− MEFs were depleted of serum for 8 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (F) Overexpression of TSC1 and TSC2 has little effect on forskolin-dependent mTORC1 activation. HEK293 cells were transfected with control or recombinant TSC1/2. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (G) Overexpression of the AMPKα1 dominant negative form does not affect forskolin-dependent mTORC1 activation. HEK293 cells were transfected with the control or recombinant form of dominant-negative AMPKα1. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. DN, dominant negative. (H) Inhibition of MAPK does not affect forskolin-dependent mTORC1 activation. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing PD98059 for 30 min and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (I) Inhibition of PKA does not affect forskolin-dependent mTORC1 activation. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing H89 for 45 min and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments.
Figure Legend Snippet: cAMP activates mTORC1 signaling via Rheb. (A) cAMP activates mTORC1 signaling in a rapamycin-dependent manner. HEK293 cells were incubated with 10% fetal bovine serum for 36 h. After 24 h of serum deprivation, the cells were preincubated with or without serum-free medium containing 10 nM rapamycin for 45 min and were then treated with 10 μM forskolin for 5 min. The cells were lysed with buffer containing 0.5% CHAPS. Equal amounts of total cell lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (B) Forskolin activates mTORC1 in a dose-dependent manner. After 24 h of serum deprivation, HEK293 cells were treated with the indicated concentrations of forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (C) cAMP-mediated mTORC1 activation is dependent on Rheb. HEK293 cells were transfected with either the control or Rheb shRNA by using Lipofectamine. After 24 h, cells were depleted of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were immunoprecipitated (IP) with anti-Rheb antibodies. The immunoprecipitates and lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. CTL, cytotoxic T lymphocytes. (D) The level of cAMP-mediated mTORC1 activation is decreased by a Rheb dominant negative mutant. HEK293 cells were transfected with either a GFP vector or GFP-Rheb D60I by using Lipofectamine. After 24 h, cells were depleted of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (E) cAMP-mediated mTORC1 activation is not affected in TSC1-deficient cells. TSC1 +/+ and TSC1 −/− MEFs were depleted of serum for 8 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (F) Overexpression of TSC1 and TSC2 has little effect on forskolin-dependent mTORC1 activation. HEK293 cells were transfected with control or recombinant TSC1/2. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of three independent experiments. (G) Overexpression of the AMPKα1 dominant negative form does not affect forskolin-dependent mTORC1 activation. HEK293 cells were transfected with the control or recombinant form of dominant-negative AMPKα1. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. DN, dominant negative. (H) Inhibition of MAPK does not affect forskolin-dependent mTORC1 activation. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing PD98059 for 30 min and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (I) Inhibition of PKA does not affect forskolin-dependent mTORC1 activation. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing H89 for 45 min and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments.

Techniques Used: Incubation, SDS Page, Activation Assay, Transfection, shRNA, Immunoprecipitation, CTL Assay, Dominant Negative Mutation, Plasmid Preparation, Over Expression, Recombinant, Inhibition

cAMP-mediated dissociation of PDE4D from Rheb enhances the formation of the Rheb-mTOR complex. (A) Schematic diagram of the GST-PDE4D fragments. (B) Rheb interacts directly with the catalytic domain of PDE4D. Each of the GST-PDE4D fragments was incubated with purified His-Rheb. After performing the GST pulldown assay, bound Rheb was analyzed by anti-Rheb antibodies, and the amounts of the GST-PDE4D fragments are shown by Ponceau S staining. The results shown are representative of two independent experiments. (C) cAMP, a substrate of PDE4D, specifically disrupts the interaction between Rheb and PDE4D. GST, GST-F2 wt, or D556A mutant fragments were incubated with His-Rheb in the presence of the indicated concentrations of cAMP or cGMP. Bound Rheb was analyzed by anti-Rheb antibodies, and the amounts of GST-F2 are shown by Ponceau S staining. The results shown are representative of two independent experiments. (D) PDE4D5 dissociates from Rheb with increased cellular cAMP levels. HEK293 cells were treated with 10 μM forskolin for the indicated times. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (E) PKA has no effect on the dynamic interaction between PDE4D5 and Rheb. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing 30 μM H89 for 45 min and then treated with 10 μM forskolin for 5 min. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (F) cAMP specifically decreases the interaction between Rheb and PDE4D5. HEK293 cells were treated with 10 nM insulin, 10 μM isoproterenol, or 10 μM forskolin for 5 min. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments.
Figure Legend Snippet: cAMP-mediated dissociation of PDE4D from Rheb enhances the formation of the Rheb-mTOR complex. (A) Schematic diagram of the GST-PDE4D fragments. (B) Rheb interacts directly with the catalytic domain of PDE4D. Each of the GST-PDE4D fragments was incubated with purified His-Rheb. After performing the GST pulldown assay, bound Rheb was analyzed by anti-Rheb antibodies, and the amounts of the GST-PDE4D fragments are shown by Ponceau S staining. The results shown are representative of two independent experiments. (C) cAMP, a substrate of PDE4D, specifically disrupts the interaction between Rheb and PDE4D. GST, GST-F2 wt, or D556A mutant fragments were incubated with His-Rheb in the presence of the indicated concentrations of cAMP or cGMP. Bound Rheb was analyzed by anti-Rheb antibodies, and the amounts of GST-F2 are shown by Ponceau S staining. The results shown are representative of two independent experiments. (D) PDE4D5 dissociates from Rheb with increased cellular cAMP levels. HEK293 cells were treated with 10 μM forskolin for the indicated times. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (E) PKA has no effect on the dynamic interaction between PDE4D5 and Rheb. After 24 h of serum deprivation, HEK293 cells were preincubated with or without serum-free medium containing 30 μM H89 for 45 min and then treated with 10 μM forskolin for 5 min. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (F) cAMP specifically decreases the interaction between Rheb and PDE4D5. HEK293 cells were treated with 10 nM insulin, 10 μM isoproterenol, or 10 μM forskolin for 5 min. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments.

Techniques Used: Incubation, Purification, GST Pulldown Assay, Staining, Mutagenesis, Immunoprecipitation, SDS Page

2) Product Images from "TRPA1 is required for TGF-β signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma"

Article Title: TRPA1 is required for TGF-β signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma

Journal: Laboratory investigation; a journal of technical methods and pathology

doi: 10.1038/labinvest.2014.85

Western blot analysis of Smad3, p38 MAPK, ERK, and JNK signaling upon exposure to TGF- β 1. Adding transforming growth factor- β 1 (TGF- β 1) to the culture medium upregulated the phosphorylation of Smad3 in 15 min, and even at 300 min phospho-Smad3 was detected in cultured ocular fibroblasts. Similarly, adding TGF- β 1 to the culture medium upregulated the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK) in 30 min, and phosphor-p38 MAPK and phospho-ERK were maintained at 300 min. Phosphorylation of c-Jun N-terminal kinase (JNK) was seen in 15 min, and even at 300 min phosphor-JNK was detected in cultured ocular fibroblasts. A loss of TRPA1 in the cells blocked the TGF- β 1-induced phosphorylation of Smad3, p38 MAPK, ERK, and JNK.
Figure Legend Snippet: Western blot analysis of Smad3, p38 MAPK, ERK, and JNK signaling upon exposure to TGF- β 1. Adding transforming growth factor- β 1 (TGF- β 1) to the culture medium upregulated the phosphorylation of Smad3 in 15 min, and even at 300 min phospho-Smad3 was detected in cultured ocular fibroblasts. Similarly, adding TGF- β 1 to the culture medium upregulated the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK) in 30 min, and phosphor-p38 MAPK and phospho-ERK were maintained at 300 min. Phosphorylation of c-Jun N-terminal kinase (JNK) was seen in 15 min, and even at 300 min phosphor-JNK was detected in cultured ocular fibroblasts. A loss of TRPA1 in the cells blocked the TGF- β 1-induced phosphorylation of Smad3, p38 MAPK, ERK, and JNK.

Techniques Used: Western Blot, Cell Culture

3) Product Images from "Human Immunodeficiency Virus Infection Alters Tumor Necrosis Factor Alpha Production via Toll-Like Receptor-Dependent Pathways in Alveolar Macrophages and U1 Cells "

Article Title: Human Immunodeficiency Virus Infection Alters Tumor Necrosis Factor Alpha Production via Toll-Like Receptor-Dependent Pathways in Alveolar Macrophages and U1 Cells

Journal: Journal of Virology

doi: 10.1128/JVI.00362-08

TNF-α induction by TLR ligands in U937 cells (solid bars) and HIV-1-infected U1 cells (open bars). Cells were differentiated with 10 nM PMA for 24 h, washed, and then incubated with Pam 3 Cys (PAM) (A and C) or LPS (ng/ml) (B and D) for 24 h. TNF-α protein (A and B) was measured by ELISA, and gene expression (C and D) was measured by real-time PCR. ERK phosphorylation was blocked by 45 min pretreatment with 25 μM PD98059 (PD) and then cells were treated with TLR ligand for 24 h. Each test was carried out in duplicate. Data are means plus SEMs (error bars) and are representative of three independent experiments. Values that that were significantly different ( P =
Figure Legend Snippet: TNF-α induction by TLR ligands in U937 cells (solid bars) and HIV-1-infected U1 cells (open bars). Cells were differentiated with 10 nM PMA for 24 h, washed, and then incubated with Pam 3 Cys (PAM) (A and C) or LPS (ng/ml) (B and D) for 24 h. TNF-α protein (A and B) was measured by ELISA, and gene expression (C and D) was measured by real-time PCR. ERK phosphorylation was blocked by 45 min pretreatment with 25 μM PD98059 (PD) and then cells were treated with TLR ligand for 24 h. Each test was carried out in duplicate. Data are means plus SEMs (error bars) and are representative of three independent experiments. Values that that were significantly different ( P =

Techniques Used: Infection, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

(A) Cytoplasmic protein extracts from HIV-positive (HIV+) AMs ( n = 3; patient 5 [P5] to P7) and HIV-negative (HIV−) healthy volunteers ( n = 4; P1 to P4) were analyzed by Western blotting for the presence of phosphorylated ERK (pERK) (p42/44) and control protein H3. We used densitometry and the Total Lab computer package to determine band intensities and normalized the phosphorylated ERK p42 band with control H3 (shown in graph) for no stimulation (NS) and LPS treatment. (B and C) Differentiated U937 cells (solid line) and U1 cells (dashed line) were treated with (+) either Pam 3 Cys (PAM) (10 ng/ml) (B) or LPS (100 ng/ml) (C) for 15, 30, or 60 min with (+) or without (−) 30 min pretreatment with the ERK inhibitor PD98059 (PD) (25 μM). We analyzed Western blots on whole-cell lysates by densitometry, and increases in phosphorylation of ERK were determined from ratios of normalized treatment over no stimulation (NS).
Figure Legend Snippet: (A) Cytoplasmic protein extracts from HIV-positive (HIV+) AMs ( n = 3; patient 5 [P5] to P7) and HIV-negative (HIV−) healthy volunteers ( n = 4; P1 to P4) were analyzed by Western blotting for the presence of phosphorylated ERK (pERK) (p42/44) and control protein H3. We used densitometry and the Total Lab computer package to determine band intensities and normalized the phosphorylated ERK p42 band with control H3 (shown in graph) for no stimulation (NS) and LPS treatment. (B and C) Differentiated U937 cells (solid line) and U1 cells (dashed line) were treated with (+) either Pam 3 Cys (PAM) (10 ng/ml) (B) or LPS (100 ng/ml) (C) for 15, 30, or 60 min with (+) or without (−) 30 min pretreatment with the ERK inhibitor PD98059 (PD) (25 μM). We analyzed Western blots on whole-cell lysates by densitometry, and increases in phosphorylation of ERK were determined from ratios of normalized treatment over no stimulation (NS).

Techniques Used: Affinity Magnetic Separation, Western Blot

4) Product Images from "Nerve Growth Factor Contribution via Transient Receptor Potential Vanilloid 1 to Ectopic Orofacial Pain"

Article Title: Nerve Growth Factor Contribution via Transient Receptor Potential Vanilloid 1 to Ectopic Orofacial Pain

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0481-11.2011

A–C , TrkA-positive whisker pad TG neurons: TrkA-positive TG neurons ( A ); FG-labeled TG neurons ( B ); FG-labeled TrkA-positive TG neurons ( C ). D–F , p75-positive whisker pad TG neurons: p75-positive TG neurons ( D ); FG-labeled TG neurons (
Figure Legend Snippet: A–C , TrkA-positive whisker pad TG neurons: TrkA-positive TG neurons ( A ); FG-labeled TG neurons ( B ); FG-labeled TrkA-positive TG neurons ( C ). D–F , p75-positive whisker pad TG neurons: p75-positive TG neurons ( D ); FG-labeled TG neurons (

Techniques Used: Whisker Assay, Labeling

5) Product Images from "Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells"

Article Title: Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-20-50

Effects of cyclic stretch on myocardin binding activity and promoter activity in VSMCs. Binding activity between myocardin and SRF and genetic transcription activity at SRF binding site of myocardin promoter increase in VSMCs under 20% cyclic stretch. ( A ) EMSA showed increased binding between myocardin and SRF under 20% cyclic stretch, which was suppressed by losartan and PD98059. Exogenous addition of AngII (10 nM) without cyclic stretch also increased the binding between myocardin and SRF. ( B and C ) Luciferase reporter assay revealed 20% cyclic stretch increased transcriptional activity at SRF binding sites of myocardin promoter when compared with the myocardin mutant, which was suppressed by PD98059 and losartan. Exogenous addition of AngII (10 nM) without cyclic stretch also increased the transcriptional activity in VSMCs. * P
Figure Legend Snippet: Effects of cyclic stretch on myocardin binding activity and promoter activity in VSMCs. Binding activity between myocardin and SRF and genetic transcription activity at SRF binding site of myocardin promoter increase in VSMCs under 20% cyclic stretch. ( A ) EMSA showed increased binding between myocardin and SRF under 20% cyclic stretch, which was suppressed by losartan and PD98059. Exogenous addition of AngII (10 nM) without cyclic stretch also increased the binding between myocardin and SRF. ( B and C ) Luciferase reporter assay revealed 20% cyclic stretch increased transcriptional activity at SRF binding sites of myocardin promoter when compared with the myocardin mutant, which was suppressed by PD98059 and losartan. Exogenous addition of AngII (10 nM) without cyclic stretch also increased the transcriptional activity in VSMCs. * P

Techniques Used: Binding Assay, Activity Assay, Luciferase, Reporter Assay, Mutagenesis

Protein synthesis in VSMCs increases after cyclic stretch. Incorporation of 3 H-proline into VSMCs in condition medium (CM) increased after 20% cyclic stretch and exogenous addition of AngII for 12 to 24 hrs and was suppressed by PD98059, losartan, AngII antibody, and myocardin siRNA. * P
Figure Legend Snippet: Protein synthesis in VSMCs increases after cyclic stretch. Incorporation of 3 H-proline into VSMCs in condition medium (CM) increased after 20% cyclic stretch and exogenous addition of AngII for 12 to 24 hrs and was suppressed by PD98059, losartan, AngII antibody, and myocardin siRNA. * P

Techniques Used:

6) Product Images from "Difference in the regulation of IL-8 expression induced by uropathogenic E. coli between two kinds of urinary tract epithelial cells"

Article Title: Difference in the regulation of IL-8 expression induced by uropathogenic E. coli between two kinds of urinary tract epithelial cells

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-16-91

MAPK signaling pathways were involved in J96-induced AP-1 activation of uroepithelial cells . Type 1 fimbriated J96 (J96-1) induced AP-1 (A) and NF-κB (C) activation in 5637 cells, and P fimbriated J96 (J96-P) induced AP-1 (B) and NF-κB (D) activation in 786-O cells were determined by TF ELISA assays in cells pretreated with vesicle (DMSO), PD98059 (PD; 30 μM), SP600125 (SP; 20 μM), or SB203580 (SB; 10 μM) individually for 1 h, or transfected with si-CL, si-p38, and then invaded with J96 for 2 h. The results are shown as mean ± SEM. * P
Figure Legend Snippet: MAPK signaling pathways were involved in J96-induced AP-1 activation of uroepithelial cells . Type 1 fimbriated J96 (J96-1) induced AP-1 (A) and NF-κB (C) activation in 5637 cells, and P fimbriated J96 (J96-P) induced AP-1 (B) and NF-κB (D) activation in 786-O cells were determined by TF ELISA assays in cells pretreated with vesicle (DMSO), PD98059 (PD; 30 μM), SP600125 (SP; 20 μM), or SB203580 (SB; 10 μM) individually for 1 h, or transfected with si-CL, si-p38, and then invaded with J96 for 2 h. The results are shown as mean ± SEM. * P

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection

Effect of MAPK inhibitors on the regulation of IL-8 expression in uroepithelail cells . (A) 5637 cells were kept as controls (CL) or invaded with type 1 fimbriated J96 (J96-1) for 4 h. (B) 786-O cells were kept as controls (CL) or invaded with P fimbriated J96 (J96-P) for 4 h. Before being kept as controls or invaded with J96, cells were pretreated with PD98059 (PD), SP600125 (SP), or SB203580 (SB) separately for 1 h. Data are normalized against 18S rRNA level and presented as fold changes in comparison to control cells (CL) and. The results are shown as mean ± SEM. * P
Figure Legend Snippet: Effect of MAPK inhibitors on the regulation of IL-8 expression in uroepithelail cells . (A) 5637 cells were kept as controls (CL) or invaded with type 1 fimbriated J96 (J96-1) for 4 h. (B) 786-O cells were kept as controls (CL) or invaded with P fimbriated J96 (J96-P) for 4 h. Before being kept as controls or invaded with J96, cells were pretreated with PD98059 (PD), SP600125 (SP), or SB203580 (SB) separately for 1 h. Data are normalized against 18S rRNA level and presented as fold changes in comparison to control cells (CL) and. The results are shown as mean ± SEM. * P

Techniques Used: Expressing

7) Product Images from "Tricin Isolated from Enzyme-Treated Zizania latifolia Extract Inhibits IgE-Mediated Allergic Reactions in RBL-2H3 Cells by Targeting the Lyn/Syk Pathway"

Article Title: Tricin Isolated from Enzyme-Treated Zizania latifolia Extract Inhibits IgE-Mediated Allergic Reactions in RBL-2H3 Cells by Targeting the Lyn/Syk Pathway

Journal: Molecules

doi: 10.3390/molecules25092084

Effects of tricin and enzyme-treated Zizania latifolia extract (ETZL) on cell viability and degranulation in IgE-stimulated rat basophilic leukemia (RBL-2H3) cells. ( A ) Cell viability; ( B ) β-hexosaminidase. RBL-2H3 cells were seeded in a 24-well plate (1 × 10 5 cells/well) overnight at 37 °C, and further incubated with anti-dinitrophenyl (DNP)-IgE (0.05 μg/mL) for 24 h. The IgE-sensitized cells were preincubated with varying concentrations of tricin (10–500 ng/mL) or ETZL (10–500 μg/mL) for 1 h, and subsequently stimulated with dinitrophenyl-human serum albumin (DNP-HSA) (0.1 μg/mL) for 4 h. All values are the mean ± S.E.M. of three independent experiments. Values of ** p
Figure Legend Snippet: Effects of tricin and enzyme-treated Zizania latifolia extract (ETZL) on cell viability and degranulation in IgE-stimulated rat basophilic leukemia (RBL-2H3) cells. ( A ) Cell viability; ( B ) β-hexosaminidase. RBL-2H3 cells were seeded in a 24-well plate (1 × 10 5 cells/well) overnight at 37 °C, and further incubated with anti-dinitrophenyl (DNP)-IgE (0.05 μg/mL) for 24 h. The IgE-sensitized cells were preincubated with varying concentrations of tricin (10–500 ng/mL) or ETZL (10–500 μg/mL) for 1 h, and subsequently stimulated with dinitrophenyl-human serum albumin (DNP-HSA) (0.1 μg/mL) for 4 h. All values are the mean ± S.E.M. of three independent experiments. Values of ** p

Techniques Used: Incubation

Inhibitory effect of tricin and ETZL on formation of leukotrienes (LT)B 4 , LTC 4 and prostaglandin E 2 (PGE 2 ) in RBL-2H3 cells stimulated by an IgE–antigen complex. ( A ) LTB 4 secretion; ( B ) LTC 4 secretion; ( C ) PGE 2 secretion. RBL-2H3 cells were seeded in a 24-well plate and cultured overnight at 37 °C; cells were subsequently washed and further incubated with anti-DNP-IgE (0.05 μg/mL) for 24 h. The cells were incubated with tricin (10–500 ng/mL) or ETZL (10–500 μg/mL) for 1 h, followed by stimulation with DNP-HSA (0.1 μg/mL) for 4 h. All values are the mean ± S.E.M. of three independent experiments. Values of * p
Figure Legend Snippet: Inhibitory effect of tricin and ETZL on formation of leukotrienes (LT)B 4 , LTC 4 and prostaglandin E 2 (PGE 2 ) in RBL-2H3 cells stimulated by an IgE–antigen complex. ( A ) LTB 4 secretion; ( B ) LTC 4 secretion; ( C ) PGE 2 secretion. RBL-2H3 cells were seeded in a 24-well plate and cultured overnight at 37 °C; cells were subsequently washed and further incubated with anti-DNP-IgE (0.05 μg/mL) for 24 h. The cells were incubated with tricin (10–500 ng/mL) or ETZL (10–500 μg/mL) for 1 h, followed by stimulation with DNP-HSA (0.1 μg/mL) for 4 h. All values are the mean ± S.E.M. of three independent experiments. Values of * p

Techniques Used: Cell Culture, Incubation

Inhibitory effect of tricin and ETZL on phosphorylation of proteins in the FcεRI signaling cascade in RBL-2H3 cells stimulated by an IgE–antigen complex. RBL-2H3 cells were seeded overnight in a 24-well plate at 37 °C; cultured cells were washed and further incubated with anti-DNP-IgE for 24 h. The cells were then incubated with varying concentrations of ( A , C ) tricin (10–500 ng/mL) or ( B , D ) ETZL (10–500 μg/mL) for 1 h, and subsequently stimulated by DNP-HSA for 4 h. All values are the mean ± S.E.M. of three independent experiments. Values of * p
Figure Legend Snippet: Inhibitory effect of tricin and ETZL on phosphorylation of proteins in the FcεRI signaling cascade in RBL-2H3 cells stimulated by an IgE–antigen complex. RBL-2H3 cells were seeded overnight in a 24-well plate at 37 °C; cultured cells were washed and further incubated with anti-DNP-IgE for 24 h. The cells were then incubated with varying concentrations of ( A , C ) tricin (10–500 ng/mL) or ( B , D ) ETZL (10–500 μg/mL) for 1 h, and subsequently stimulated by DNP-HSA for 4 h. All values are the mean ± S.E.M. of three independent experiments. Values of * p

Techniques Used: Cell Culture, Incubation

8) Product Images from "ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor ?"

Article Title: ERBB Receptor Activation Is Required for Profibrotic Responses to Transforming Growth Factor ?

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-0232

Activation of the ERBB axis by TGF-β is SMAD dependent. A, AKR-2B cells stably expressing shRNA targeting Smad2 or Smad3 were treated with TGF-β (10 ng/mL) for 6 or 12 h and harvested for total RNA extraction. Untransduced AKR-2B cells (Untr) and cells transduced with nontargeting sequences (NT-Ctrl) were used as controls. Samples were subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Rpl13a was used as an internal control. B, cells were treated as in A, and total proteins (500 μg) were subjected to immunoprecipitation (IP) using ERBB1-specific antibodies. Immunoprecipitates were analyzed by Western blotting using phosphotyrosine (pY)–specific antibodies. Membranes were stripped and reprobed with ERBB1 antibodies. Equivalent protein aliquots were subjected to Western blot analysis using antibodies specific for ERBB1, phosphorylated SMAD2 (p-SMAD2), and phosphorylated SMAD3 (p-SMAD3). SMAD2 and SMAD3 antibodies were used to determine silencing efficiency for both genes. GAPDH served as an internal control. C, murine embryonic fibroblast cells, lacking expression of Smad2 ( Smad2 −/− ) as well as the WT counterpart, were treated with TGF-β (10 ng/mL), harvested at various time points, and subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Gapdh was used as an internal control. D, cells were treated with TGF-β as in C, and total proteins (50 μg) were analyzed by Western blotting using antibodies specific for phosphorylated SMAD2 (p-SMAD2), phosphorylated SMAD3 (p-SMAD3), or total SMAD2/3.
Figure Legend Snippet: Activation of the ERBB axis by TGF-β is SMAD dependent. A, AKR-2B cells stably expressing shRNA targeting Smad2 or Smad3 were treated with TGF-β (10 ng/mL) for 6 or 12 h and harvested for total RNA extraction. Untransduced AKR-2B cells (Untr) and cells transduced with nontargeting sequences (NT-Ctrl) were used as controls. Samples were subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Rpl13a was used as an internal control. B, cells were treated as in A, and total proteins (500 μg) were subjected to immunoprecipitation (IP) using ERBB1-specific antibodies. Immunoprecipitates were analyzed by Western blotting using phosphotyrosine (pY)–specific antibodies. Membranes were stripped and reprobed with ERBB1 antibodies. Equivalent protein aliquots were subjected to Western blot analysis using antibodies specific for ERBB1, phosphorylated SMAD2 (p-SMAD2), and phosphorylated SMAD3 (p-SMAD3). SMAD2 and SMAD3 antibodies were used to determine silencing efficiency for both genes. GAPDH served as an internal control. C, murine embryonic fibroblast cells, lacking expression of Smad2 ( Smad2 −/− ) as well as the WT counterpart, were treated with TGF-β (10 ng/mL), harvested at various time points, and subjected to RT-PCR analysis using primers specific for Areg, Ereg , or Hbegf. Gapdh was used as an internal control. D, cells were treated with TGF-β as in C, and total proteins (50 μg) were analyzed by Western blotting using antibodies specific for phosphorylated SMAD2 (p-SMAD2), phosphorylated SMAD3 (p-SMAD3), or total SMAD2/3.

Techniques Used: Activation Assay, Stable Transfection, Expressing, shRNA, RNA Extraction, Transduction, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot

9) Product Images from "Selective cyclooxygenase inhibition by SC-560 improves hepatopulmonary syndrome in cirrhotic rats"

Article Title: Selective cyclooxygenase inhibition by SC-560 improves hepatopulmonary syndrome in cirrhotic rats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0179809

Pulmonary protein expressions of VEGF, RhoA, VEGFR-1, VEGFR-2, Akt, PI3K and ERK. SC-560 and celecoxib significantly down-regulated VEGF and RhoA expressions (6A). The phosphorylated VEGFR-2 expression was significantly up-regulated by celecoxib (6B). The phosphorylated Akt and ERK (42) protein levels were also significantly up-regulated by celecoxib (6C D). The PI3K and phosphorylated ERK (44) expressions were not significantly influenced by celecoxib (6C D).
Figure Legend Snippet: Pulmonary protein expressions of VEGF, RhoA, VEGFR-1, VEGFR-2, Akt, PI3K and ERK. SC-560 and celecoxib significantly down-regulated VEGF and RhoA expressions (6A). The phosphorylated VEGFR-2 expression was significantly up-regulated by celecoxib (6B). The phosphorylated Akt and ERK (42) protein levels were also significantly up-regulated by celecoxib (6C D). The PI3K and phosphorylated ERK (44) expressions were not significantly influenced by celecoxib (6C D).

Techniques Used: Expressing

10) Product Images from "Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells"

Article Title: Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0149269

Scavenge of NO with C-PTIO (A) and inhibition of ERK signal pathway with U0126 (B) recover the same levels of fibronectin (Fb) and laminin β-1(LMN β-1) in the cells treated with control (Ctrl), OGD, tPA, and OGD+tPA (O+t). Upper panel: representative immunoblots, β-actin served as loading control; Bottom panel: band intensity was quantitated after normalization to β-actin and the data were expressed as mean ± SD, ANOVA, N = 4.
Figure Legend Snippet: Scavenge of NO with C-PTIO (A) and inhibition of ERK signal pathway with U0126 (B) recover the same levels of fibronectin (Fb) and laminin β-1(LMN β-1) in the cells treated with control (Ctrl), OGD, tPA, and OGD+tPA (O+t). Upper panel: representative immunoblots, β-actin served as loading control; Bottom panel: band intensity was quantitated after normalization to β-actin and the data were expressed as mean ± SD, ANOVA, N = 4.

Techniques Used: Inhibition, Western Blot

11) Product Images from "Exendin-4 Suppresses Src Activation and Reactive Oxygen Species Production in Diabetic Goto-Kakizaki Rat Islets in an Epac-Dependent Manner"

Article Title: Exendin-4 Suppresses Src Activation and Reactive Oxygen Species Production in Diabetic Goto-Kakizaki Rat Islets in an Epac-Dependent Manner

Journal: Diabetes

doi: 10.2337/db10-0021

Comparison of expression of Src between fresh Wistar and GK islets. Fresh islets were lysated and subjected to immunoblot analyses. Blots (50 μg of protein) were probed with anti–phospho-Src (Tyr 416 ), anti–phospho-Src (Tyr 527 ), anti-Src, or anti-Csk. The same blots were stripped and reprobed with anti–β-actin, respectively. Intensities of the bands were quantified with densitometric imager. The bar graphs are expressed relative to Wistar islet value corrected by β-actin level (means ± SE). * P
Figure Legend Snippet: Comparison of expression of Src between fresh Wistar and GK islets. Fresh islets were lysated and subjected to immunoblot analyses. Blots (50 μg of protein) were probed with anti–phospho-Src (Tyr 416 ), anti–phospho-Src (Tyr 527 ), anti-Src, or anti-Csk. The same blots were stripped and reprobed with anti–β-actin, respectively. Intensities of the bands were quantified with densitometric imager. The bar graphs are expressed relative to Wistar islet value corrected by β-actin level (means ± SE). * P

Techniques Used: Expressing

12) Product Images from "Fibronectin is essential for survival but is dispensable for proliferation of hepatocytes in acute liver injury"

Article Title: Fibronectin is essential for survival but is dispensable for proliferation of hepatocytes in acute liver injury

Journal: Hepatology (Baltimore, Md.)

doi: 10.1002/hep.25624

Analysis of intracellular signalings following liver injury ( A ) Western blot analysis of phospho-ERK (pERK), total ERK, phospho-Akt (pAkt), total Akt, p53, and HSC70 (loading control) in control and mutant livers. Pooled samples from 3 mouse livers from each strain were used for the analysis. Band intensity was measured by densitometry, and each pERK and pAkt intensity was normalized to total ERK and total Akt, respectively; each p53 intensity was normalized to HSC70. Then the intensity at 0hr in the control liver was set to 1. Each intensity is shown relative to the control value. ( B ) Immunostaining for pERK (in brown) at 36 and 48hrs post injury Sections were counterstained with hematoxylin. CV, central vein. Bar=50μm. Inset: Double immunofluorescence staining for pERK (in red) and albumin (in green) in control and fibronectin-null livers at 36hrs post injury. Arrowheads indicate double-positive hepatocytes (orange to yellow color). Bar=50μm. ( C ) Analysis of pERK-positive hepatocytes. Data are means±S.D. (cells/field; field=0.14mm 2 [ n =5 for each group]). **, P
Figure Legend Snippet: Analysis of intracellular signalings following liver injury ( A ) Western blot analysis of phospho-ERK (pERK), total ERK, phospho-Akt (pAkt), total Akt, p53, and HSC70 (loading control) in control and mutant livers. Pooled samples from 3 mouse livers from each strain were used for the analysis. Band intensity was measured by densitometry, and each pERK and pAkt intensity was normalized to total ERK and total Akt, respectively; each p53 intensity was normalized to HSC70. Then the intensity at 0hr in the control liver was set to 1. Each intensity is shown relative to the control value. ( B ) Immunostaining for pERK (in brown) at 36 and 48hrs post injury Sections were counterstained with hematoxylin. CV, central vein. Bar=50μm. Inset: Double immunofluorescence staining for pERK (in red) and albumin (in green) in control and fibronectin-null livers at 36hrs post injury. Arrowheads indicate double-positive hepatocytes (orange to yellow color). Bar=50μm. ( C ) Analysis of pERK-positive hepatocytes. Data are means±S.D. (cells/field; field=0.14mm 2 [ n =5 for each group]). **, P

Techniques Used: Western Blot, Mutagenesis, Immunostaining, Double Immunofluorescence Staining

Fibrin(ogen) alone forms the initial matrix without fibronectin in response to CCl 4 -induced acute liver injury ( A ) Western blot analysis of fibronectin and fibrinogen in control and fibronectin-null livers at 0, 6, 12, 36 and 72hrs after injury under reducing conditions. Pooled samples from 3 mouse livers from each strain were used for the analysis. Band intensity was measured by densitometry and normalized to HSC70 (loading control), then the intensity of the control liver at 0hr was set to 1. Each intensity is shown relative to the control value. ( B ) Time-course of fibronectin and fibrin(ogen) deposition in control and Fn-null livers at 12, 36, and 72hrs after injury. Double immunofluorescence staining for fibronectin (in red) and fibrin(ogen) (in green), and the merged images. CV, central vein. Bar=50μm.
Figure Legend Snippet: Fibrin(ogen) alone forms the initial matrix without fibronectin in response to CCl 4 -induced acute liver injury ( A ) Western blot analysis of fibronectin and fibrinogen in control and fibronectin-null livers at 0, 6, 12, 36 and 72hrs after injury under reducing conditions. Pooled samples from 3 mouse livers from each strain were used for the analysis. Band intensity was measured by densitometry and normalized to HSC70 (loading control), then the intensity of the control liver at 0hr was set to 1. Each intensity is shown relative to the control value. ( B ) Time-course of fibronectin and fibrin(ogen) deposition in control and Fn-null livers at 12, 36, and 72hrs after injury. Double immunofluorescence staining for fibronectin (in red) and fibrin(ogen) (in green), and the merged images. CV, central vein. Bar=50μm.

Techniques Used: Western Blot, Double Immunofluorescence Staining

The lack of fibronectin significantly diminishes survival signals for hepatocytes, mediated by Bcl-xL ( A ) Immunostaining for cleaved caspase 3 (in brown) at 36hrs after injury. Sections were counterstained with hematoxylin. CV, central vein. Bar=50μm. ( B ) Analysis of cleaved caspase 3-positive cells. Data are means±S.D. (cells/field; field=0.14mm 2 [ n =5 for each group]). *, P
Figure Legend Snippet: The lack of fibronectin significantly diminishes survival signals for hepatocytes, mediated by Bcl-xL ( A ) Immunostaining for cleaved caspase 3 (in brown) at 36hrs after injury. Sections were counterstained with hematoxylin. CV, central vein. Bar=50μm. ( B ) Analysis of cleaved caspase 3-positive cells. Data are means±S.D. (cells/field; field=0.14mm 2 [ n =5 for each group]). *, P

Techniques Used: Immunostaining

Analysis of liver damage following acute injury ( A ) Serum ALT levels in control and fibronectin-null mice post injury. Data are means±S.D. ( n =10 for each group). ( B ) Upper panel: Time course of the quantification of damaged areas using Periodic Acid-Schiff (PAS)-stained tissue sections. Data are the means±S.D. ( n =5 for each group). **, P
Figure Legend Snippet: Analysis of liver damage following acute injury ( A ) Serum ALT levels in control and fibronectin-null mice post injury. Data are means±S.D. ( n =10 for each group). ( B ) Upper panel: Time course of the quantification of damaged areas using Periodic Acid-Schiff (PAS)-stained tissue sections. Data are the means±S.D. ( n =5 for each group). **, P

Techniques Used: Mouse Assay, Staining

Fibronectin deficiency significantly increases hepatocyte proliferation post injury ( A ) Immunostaining for Ki67 (in brown) at 36 and 72hrs post injury. Sections were counterstained with hematoxylin. Arrowheads indicate positive cells. CV, central vein. Bar=50μm. ( B ) Upper panels: Triple immunofluorescence staining for Ki67 (in red), albumin (in green) and DAPI (in blue) at 36hrs post injury. Arrowheads indicate Ki67 and albumin double-positive hepatocytes. Bar=25μm. Lower panel: Analysis of Ki67-positive hepatocytes. Data are the means±S.D. (cells/field; field=0.14mm 2 [ n =5 for each group]). **, P
Figure Legend Snippet: Fibronectin deficiency significantly increases hepatocyte proliferation post injury ( A ) Immunostaining for Ki67 (in brown) at 36 and 72hrs post injury. Sections were counterstained with hematoxylin. Arrowheads indicate positive cells. CV, central vein. Bar=50μm. ( B ) Upper panels: Triple immunofluorescence staining for Ki67 (in red), albumin (in green) and DAPI (in blue) at 36hrs post injury. Arrowheads indicate Ki67 and albumin double-positive hepatocytes. Bar=25μm. Lower panel: Analysis of Ki67-positive hepatocytes. Data are the means±S.D. (cells/field; field=0.14mm 2 [ n =5 for each group]). **, P

Techniques Used: Immunostaining, Immunofluorescence, Staining

Fibronectin deficiency significantly upregulates liver HGF levels post injury ( A ) Double immunofluorescence staining for cytoglobin (in red)/DAPI (in blue) and α-SMA (in red)/DAPI (in blue) at 36 and 72hrs post injury. CV, central vein. Bar=50μm. ( B ) Hepatic HGF levels at 36hrs post injury. Data are means±S.D. ( n =4 for each group). **, P
Figure Legend Snippet: Fibronectin deficiency significantly upregulates liver HGF levels post injury ( A ) Double immunofluorescence staining for cytoglobin (in red)/DAPI (in blue) and α-SMA (in red)/DAPI (in blue) at 36 and 72hrs post injury. CV, central vein. Bar=50μm. ( B ) Hepatic HGF levels at 36hrs post injury. Data are means±S.D. ( n =4 for each group). **, P

Techniques Used: Double Immunofluorescence Staining

13) Product Images from "Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells"

Article Title: Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

Journal: Breast Cancer Research

doi: 10.1186/bcr1392

Disrupted apico-basal polarity of IGFIR structures. Equatorial confocal sections of 20-day control (pCLXSN, panels a, c, e) and IGFIR structures (panels b, d, f) labeled with antibodies to laminin V (red; panels a, b), E-cadherin (red; panels c, d) and Golgi matrix protein of 130 kDa (GM130; green; panels e, f) and the nuclear counterstain TO-PRO-3 (blue). Arrows indicate localization of E-cadherin at cell-cell junctions (panel d) and apical localization of GM130 in control structures (panel e). Arrowheads show sites of punctate E-cadherin labeling (panel d). Scale bars = 50 μm.
Figure Legend Snippet: Disrupted apico-basal polarity of IGFIR structures. Equatorial confocal sections of 20-day control (pCLXSN, panels a, c, e) and IGFIR structures (panels b, d, f) labeled with antibodies to laminin V (red; panels a, b), E-cadherin (red; panels c, d) and Golgi matrix protein of 130 kDa (GM130; green; panels e, f) and the nuclear counterstain TO-PRO-3 (blue). Arrows indicate localization of E-cadherin at cell-cell junctions (panel d) and apical localization of GM130 in control structures (panel e). Arrowheads show sites of punctate E-cadherin labeling (panel d). Scale bars = 50 μm.

Techniques Used: Labeling

14) Product Images from "Cyclic AMP Controls mTOR through Regulation of the Dynamic Interaction between Rheb and Phosphodiesterase 4D ▿"

Article Title: Cyclic AMP Controls mTOR through Regulation of the Dynamic Interaction between Rheb and Phosphodiesterase 4D ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00217-10

PDE4D has a negative effect on mTORC1 kinase activity. (A) cAMP enhances the interaction between Rheb and mTOR. HEK293 cells were treated with 10 μM forskolin or 10 μM isoproterenol for 5 min. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (B) The PDE4D F2 fragment inhibits mTORC1 kinase activity. Myc-mTOR and FLAG-Raptor were transfected into HEK293 cells. After immunoprecipitation with anti-FLAG antibodies, immunoprecipitates and GTPγS-loaded GST-Rheb were incubated in the absence or presence of the F2 fragment at 37°C for 10 min. Proteins were resolved by SDS-PAGE, and the phosphorylation of 4EBP1 on Thr37/46 was analyzed by Western blotting. The bar graph at the bottom shows the quantification of 4EBP1 phosphorylation. The results shown are representative of three independent experiments. (C) mTORC1 activity inhibited by the F2 fragment is restored by cAMP but not cGMP. Myc-mTOR and FLAG-Raptor were transfected into HEK293 cells. After immunoprecipitation with anti-FLAG antibodies, immunoprecipitates, GTPγS-loaded GST-Rheb, and the F2 fragment were incubated in the absence or presence of cAMP or cGMP at 37°C for 10 min. Proteins were resolved by SDS-PAGE, and the phosphorylation of 4EBP1 on Thr37/46 was analyzed by Western blotting. The bar graph at the bottom shows the quantification of 4EBP1 phosphorylation. The results shown are representative of two independent experiments. (D) The PDE4D F2 fragment inhibits mTORC1 activity. HEK293 cells were transfected with the indicated constructs. After 36 h, the cells were lysed, subjected to SDS-PAGE, and immunoblotted with the respective antibodies. The bar graph at the bottom shows the quantification of S6K1 phosphorylation. The results shown are representative of three independent experiments. (E) The PDE4D F2 fragment inhibits mTORC1 activity. HEK293 cells were transfected with the indicated constructs and Myc-S6K1. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The bar graph at the bottom shows the quantification of S6K1 phosphorylation. The results shown are representative of two independent experiments. (F) PDE4D knockdown does not affect insulin-mediated mTORC1 activity. HEK293 cells were transfected with either control or PDE4D siRNA. After 24 h, cells were deprived of serum for 24 h and then treated with 20 nM insulin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (G) PDE4D5 wt overexpression does not affect insulin-mediated mTORC1 activity. HEK293 cells were transfected with either the control or wt PDE4D5 constructs. After 24 h, cells were deprived of serum for 24 h and then treated with 20 nM insulin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (H) PDE5A does not affect mTORC1 activity. HEK 293 cells were transfected with the indicated constructs. After 36 h, the cells were lysed, subjected to SDS-PAGE, and immunoblotted with the respective antibodies. 4D5, PDE4D5; m5A, mPDE5A.
Figure Legend Snippet: PDE4D has a negative effect on mTORC1 kinase activity. (A) cAMP enhances the interaction between Rheb and mTOR. HEK293 cells were treated with 10 μM forskolin or 10 μM isoproterenol for 5 min. The cell lysates were immunoprecipitated with anti-Rheb antibodies. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The results shown are representative of two independent experiments. (B) The PDE4D F2 fragment inhibits mTORC1 kinase activity. Myc-mTOR and FLAG-Raptor were transfected into HEK293 cells. After immunoprecipitation with anti-FLAG antibodies, immunoprecipitates and GTPγS-loaded GST-Rheb were incubated in the absence or presence of the F2 fragment at 37°C for 10 min. Proteins were resolved by SDS-PAGE, and the phosphorylation of 4EBP1 on Thr37/46 was analyzed by Western blotting. The bar graph at the bottom shows the quantification of 4EBP1 phosphorylation. The results shown are representative of three independent experiments. (C) mTORC1 activity inhibited by the F2 fragment is restored by cAMP but not cGMP. Myc-mTOR and FLAG-Raptor were transfected into HEK293 cells. After immunoprecipitation with anti-FLAG antibodies, immunoprecipitates, GTPγS-loaded GST-Rheb, and the F2 fragment were incubated in the absence or presence of cAMP or cGMP at 37°C for 10 min. Proteins were resolved by SDS-PAGE, and the phosphorylation of 4EBP1 on Thr37/46 was analyzed by Western blotting. The bar graph at the bottom shows the quantification of 4EBP1 phosphorylation. The results shown are representative of two independent experiments. (D) The PDE4D F2 fragment inhibits mTORC1 activity. HEK293 cells were transfected with the indicated constructs. After 36 h, the cells were lysed, subjected to SDS-PAGE, and immunoblotted with the respective antibodies. The bar graph at the bottom shows the quantification of S6K1 phosphorylation. The results shown are representative of three independent experiments. (E) The PDE4D F2 fragment inhibits mTORC1 activity. HEK293 cells were transfected with the indicated constructs and Myc-S6K1. After 24 h, cells were deprived of serum for 24 h and then treated with 10 μM forskolin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. The bar graph at the bottom shows the quantification of S6K1 phosphorylation. The results shown are representative of two independent experiments. (F) PDE4D knockdown does not affect insulin-mediated mTORC1 activity. HEK293 cells were transfected with either control or PDE4D siRNA. After 24 h, cells were deprived of serum for 24 h and then treated with 20 nM insulin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (G) PDE4D5 wt overexpression does not affect insulin-mediated mTORC1 activity. HEK293 cells were transfected with either the control or wt PDE4D5 constructs. After 24 h, cells were deprived of serum for 24 h and then treated with 20 nM insulin for 5 min. The lysates were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (H) PDE5A does not affect mTORC1 activity. HEK 293 cells were transfected with the indicated constructs. After 36 h, the cells were lysed, subjected to SDS-PAGE, and immunoblotted with the respective antibodies. 4D5, PDE4D5; m5A, mPDE5A.

Techniques Used: Activity Assay, Immunoprecipitation, SDS Page, Transfection, Incubation, Western Blot, Construct, Over Expression

15) Product Images from "Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells"

Article Title: Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimensional culture model of breast epithelial cells

Journal: Breast Cancer Research

doi: 10.1186/bcr1392

Induction of Akt signaling in IGFIR structures. Equatorial confocal sections of 34-day control (pCLXSN, panels a, c) and IGFIR acinar structures (panels c, d) labeled with phosphospecific antibodies to Akt- [S473] (green; panels a, b) and mammalian target of rapamycin (mTOR)- [S2448] (green; panels c, d) and the nuclear counterstain TO-PRO-3 (blue). Scale bars = 50 μm.
Figure Legend Snippet: Induction of Akt signaling in IGFIR structures. Equatorial confocal sections of 34-day control (pCLXSN, panels a, c) and IGFIR acinar structures (panels c, d) labeled with phosphospecific antibodies to Akt- [S473] (green; panels a, b) and mammalian target of rapamycin (mTOR)- [S2448] (green; panels c, d) and the nuclear counterstain TO-PRO-3 (blue). Scale bars = 50 μm.

Techniques Used: Labeling

Inhibition of PI3K or MEK but not mTOR disrupts IGFIR acinar development. (a) Confocal sections of 20-day control (pCLXSN, panels a, d, g) and IGFIR (panels b, c, e, f, h, i) acinar structures grown in the presence of the MEK inhibitor, UO126 (10 μm; panels a-c), the PI3K inhibitor LY294002 (50 μm; panels d-f) and the mTOR inhibitor rapamycin (40 nM; panels g-i). Fixed structures were labeled with the nuclear counterstain TO-PRO-3 (blue) and with antibodies to Ki-67 (green; panels a, b, d, e, g, h) or cleaved caspase-3 (green; panels c, f, i). Scale bars = 50 μm. The arrow in panel b indicates Ki-67-labeled cells. (b) Whole-cell lysates of 16d IGFIR acinar structures were isolated after overnight serum starvation followed by 30 minute pretreatment with DMSO (vehicle) or 40 nM rapamycin and subsequent 30-minute incubation with 10 μg/ml insulin. Lysates were immunoprecipitated with antibodies to p70S6K, separated by 12.5% SDS-PAGE, transferred to PVDF and immunoblotted with antibodies to p70S6K or phosphospecific antibodies to Thr389 of p70S6K as indicated.
Figure Legend Snippet: Inhibition of PI3K or MEK but not mTOR disrupts IGFIR acinar development. (a) Confocal sections of 20-day control (pCLXSN, panels a, d, g) and IGFIR (panels b, c, e, f, h, i) acinar structures grown in the presence of the MEK inhibitor, UO126 (10 μm; panels a-c), the PI3K inhibitor LY294002 (50 μm; panels d-f) and the mTOR inhibitor rapamycin (40 nM; panels g-i). Fixed structures were labeled with the nuclear counterstain TO-PRO-3 (blue) and with antibodies to Ki-67 (green; panels a, b, d, e, g, h) or cleaved caspase-3 (green; panels c, f, i). Scale bars = 50 μm. The arrow in panel b indicates Ki-67-labeled cells. (b) Whole-cell lysates of 16d IGFIR acinar structures were isolated after overnight serum starvation followed by 30 minute pretreatment with DMSO (vehicle) or 40 nM rapamycin and subsequent 30-minute incubation with 10 μg/ml insulin. Lysates were immunoprecipitated with antibodies to p70S6K, separated by 12.5% SDS-PAGE, transferred to PVDF and immunoblotted with antibodies to p70S6K or phosphospecific antibodies to Thr389 of p70S6K as indicated.

Techniques Used: Inhibition, Labeling, Isolation, Incubation, Immunoprecipitation, SDS Page

16) Product Images from "ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation"

Article Title: ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr2475

Induction of caspase activities by ABL-N . MDA-MB-231 cells were treated with 20 μM ABL-N for indicated times. (a) Whole cell protein lysates were prepared and subjected to Western blot analysis for detection of cleavage of caspase-3 and caspase-9. (b) Induction of caspase activities by ABL-N in MDA-MB-231 cells. (c) Cleavage of PARP was induced by ABL-N. (d) Cells were pretreated with 50 μM of either z-VAD-fmk or z-DEVD-fmk for one hour, followed by 20 μM ABL-N for 24 hours, and caspase-3 activity, DNA fragmentation and sub-G 1 DNA contents were determined. The data are expressed as means ± SE of three separate experiments. (e) Abrogation of PARP cleavage by caspase inhibitors.
Figure Legend Snippet: Induction of caspase activities by ABL-N . MDA-MB-231 cells were treated with 20 μM ABL-N for indicated times. (a) Whole cell protein lysates were prepared and subjected to Western blot analysis for detection of cleavage of caspase-3 and caspase-9. (b) Induction of caspase activities by ABL-N in MDA-MB-231 cells. (c) Cleavage of PARP was induced by ABL-N. (d) Cells were pretreated with 50 μM of either z-VAD-fmk or z-DEVD-fmk for one hour, followed by 20 μM ABL-N for 24 hours, and caspase-3 activity, DNA fragmentation and sub-G 1 DNA contents were determined. The data are expressed as means ± SE of three separate experiments. (e) Abrogation of PARP cleavage by caspase inhibitors.

Techniques Used: Multiple Displacement Amplification, Western Blot, Activity Assay

Role of JNK in ABL-N-induced apoptosis . MDA-MB-231 cells were incubated with ABL-N (20 μM) and either SB203580 (20 μM) or SP600125 (30 μM), or a combination of both for 24 hours. (a) MDA-MB-231 cells were transfected with JNK siRNA (25 nM) or control siRNA (25 nM) for 48 hours, and cell lysates were subjected to Western blot with antibodies to JNK. (b) ABL-N-induced cell death was abrogated by inhibition of JNK using MTT assay. (c) Sub G 1 DNA content was analyzed by flow cytometry. (d) Caspase-3 activity was determined by Caspase-Glo assay and the cleavage of PARP was analyzed via Western blotting. Data are expressed as the means ± SE of three independent experiments. (e) Effect of MAP kinase inhibitors on ABL-N-induced JNK activation. (f) Effect of caspase inhibitors on JNK activation. Cells pretreated with or without z-VAD-fmk (50 μM) or z-DEVD-fmk (50 μM, one hour) were further incubated with vehicle or 20 μM ABL-N for 24 hours. Data are representative of three independent experiments.
Figure Legend Snippet: Role of JNK in ABL-N-induced apoptosis . MDA-MB-231 cells were incubated with ABL-N (20 μM) and either SB203580 (20 μM) or SP600125 (30 μM), or a combination of both for 24 hours. (a) MDA-MB-231 cells were transfected with JNK siRNA (25 nM) or control siRNA (25 nM) for 48 hours, and cell lysates were subjected to Western blot with antibodies to JNK. (b) ABL-N-induced cell death was abrogated by inhibition of JNK using MTT assay. (c) Sub G 1 DNA content was analyzed by flow cytometry. (d) Caspase-3 activity was determined by Caspase-Glo assay and the cleavage of PARP was analyzed via Western blotting. Data are expressed as the means ± SE of three independent experiments. (e) Effect of MAP kinase inhibitors on ABL-N-induced JNK activation. (f) Effect of caspase inhibitors on JNK activation. Cells pretreated with or without z-VAD-fmk (50 μM) or z-DEVD-fmk (50 μM, one hour) were further incubated with vehicle or 20 μM ABL-N for 24 hours. Data are representative of three independent experiments.

Techniques Used: Multiple Displacement Amplification, Incubation, Transfection, Western Blot, Inhibition, MTT Assay, Flow Cytometry, Cytometry, Activity Assay, Caspase-Glo Assay, Activation Assay

17) Product Images from "Opposing Roles of Integrin ?6A?1 and Dystroglycan in Laminin-mediated Extracellular Signal-regulated Kinase Activation"

Article Title: Opposing Roles of Integrin ?6A?1 and Dystroglycan in Laminin-mediated Extracellular Signal-regulated Kinase Activation

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E03-01-0852

ERK and MEK activation in K562 cells expressing integrin α6A or α6B variants. (A) Cell adhesion at 1 h to the indicated laminin isoforms, integrin α6 antibodies or to control antibodies (20 μg/ml) was measured colorimetrically. Each point represents the mean of triplicate wells (± SD). The same results were obtained in two different experiments. (B) K562α6A and K562α6B cells were attached to wells precoated with laminin-10/11 (10 μg/ml) for 1 h. The presence of kinases and their phosphorylation status was examined by immunoblotting as described previously. Quantitative measurements of the phosphorylation status of MEK (C) and ERK (D). Phosphorylation of MEK1/2 and ERK1/2 in cells kept in suspension were set as 100%. Triplicate experiments gave the same results.
Figure Legend Snippet: ERK and MEK activation in K562 cells expressing integrin α6A or α6B variants. (A) Cell adhesion at 1 h to the indicated laminin isoforms, integrin α6 antibodies or to control antibodies (20 μg/ml) was measured colorimetrically. Each point represents the mean of triplicate wells (± SD). The same results were obtained in two different experiments. (B) K562α6A and K562α6B cells were attached to wells precoated with laminin-10/11 (10 μg/ml) for 1 h. The presence of kinases and their phosphorylation status was examined by immunoblotting as described previously. Quantitative measurements of the phosphorylation status of MEK (C) and ERK (D). Phosphorylation of MEK1/2 and ERK1/2 in cells kept in suspension were set as 100%. Triplicate experiments gave the same results.

Techniques Used: Activation Assay, Expressing

ERK phosphorylation in WI-26 VA4 and WCCS-1 cells attached to laminin-1 or laminin-10/11. (A). Serum-starved cells were detached and kept in suspension or cultured for 60 min on plates coated with laminin-1 or -10/11 at 10 μg/ml as indicated in the figure. Total cellular protein extracts (20 μg) were subjected to Western blot analysis by using antibodies specific for the phosphorylated isoforms of ERK1/2. To evaluate loading efficiency, membranes were stripped and reprobed with antibodies recognizing ERK2 irrespective of its phosphorylation status. The positions of phospho-ERK1/2 and ERK2 are indicated. The same results were seen in more than three experiments for both cell lines. (B) WCCS-1 cells were cultured in medium containing 10% fetal calf serum for 48 h. ERK activation was then measured as in A from cells cultured for an additional 5 min in the presence or absence of basic fibroblast growth factor at 5 nM. (C) Serum-starved cells were detached and then cultured with or without the MEK-specific inhibitor PD98059 (50 μM, PD) for 30 min in suspension and then for 30 min on wells coated with laminin-10/11 at 10 μg/ml. The presence of kinases and their phosphorylated forms was detected by immunoblotting as described above. The positions of phospho-MEK1/2, phospho-ERK1/2, MEK2, and ERK1 are indicated. (D) Quantitative measurements of the phosphorylation status of the kinases. Phosphorylation of MEK1/2 and ERK1/2 in nontreated control cells attached to laminin-10/11 was set as 100%. The same results were seen in three independent experiments.
Figure Legend Snippet: ERK phosphorylation in WI-26 VA4 and WCCS-1 cells attached to laminin-1 or laminin-10/11. (A). Serum-starved cells were detached and kept in suspension or cultured for 60 min on plates coated with laminin-1 or -10/11 at 10 μg/ml as indicated in the figure. Total cellular protein extracts (20 μg) were subjected to Western blot analysis by using antibodies specific for the phosphorylated isoforms of ERK1/2. To evaluate loading efficiency, membranes were stripped and reprobed with antibodies recognizing ERK2 irrespective of its phosphorylation status. The positions of phospho-ERK1/2 and ERK2 are indicated. The same results were seen in more than three experiments for both cell lines. (B) WCCS-1 cells were cultured in medium containing 10% fetal calf serum for 48 h. ERK activation was then measured as in A from cells cultured for an additional 5 min in the presence or absence of basic fibroblast growth factor at 5 nM. (C) Serum-starved cells were detached and then cultured with or without the MEK-specific inhibitor PD98059 (50 μM, PD) for 30 min in suspension and then for 30 min on wells coated with laminin-10/11 at 10 μg/ml. The presence of kinases and their phosphorylated forms was detected by immunoblotting as described above. The positions of phospho-MEK1/2, phospho-ERK1/2, MEK2, and ERK1 are indicated. (D) Quantitative measurements of the phosphorylation status of the kinases. Phosphorylation of MEK1/2 and ERK1/2 in nontreated control cells attached to laminin-10/11 was set as 100%. The same results were seen in three independent experiments.

Techniques Used: Cell Culture, Western Blot, Activation Assay

18) Product Images from "Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia"

Article Title: Proangiogenic Effect of Metformin in Endothelial Cells Is via Upregulation of VEGFR1/2 and Their Signaling under Hyperglycemia-Hypoxia

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19010293

Effect of metformin on the activity of MAPK pathway. HUVEC were treated with euglycemia or hyperglycemia in the presence or absence of metformin for 48 h, and parallel cultures were exposed to CoCl 2 for 12 h. ( A ) Represents % of ERK1/2 phosphorylation ( B ) Effect of metformin on euglycemia, and ( C ) hyperglycemia combined with 12 h chemical hypoxia CoCl 2 . Percentage of phosphorylation indicates relative intensity of phospho-protein/total protein. The variation in protein expression levels was assessed by flow cytometry on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using paired t -test. * p
Figure Legend Snippet: Effect of metformin on the activity of MAPK pathway. HUVEC were treated with euglycemia or hyperglycemia in the presence or absence of metformin for 48 h, and parallel cultures were exposed to CoCl 2 for 12 h. ( A ) Represents % of ERK1/2 phosphorylation ( B ) Effect of metformin on euglycemia, and ( C ) hyperglycemia combined with 12 h chemical hypoxia CoCl 2 . Percentage of phosphorylation indicates relative intensity of phospho-protein/total protein. The variation in protein expression levels was assessed by flow cytometry on three independent biological replicates. Results are presented as mean ± SEM and were analyzed using paired t -test. * p

Techniques Used: Activity Assay, Expressing, Flow Cytometry, Cytometry

Comprehensive VEGF signaling network of genes and proteins involved in cell migration and survival. Metformin-treated condition is compared to metformin-untreated condition under hyperglycemia-CoCl 2 for 12 h. The network was created by IPA software rendering VEGF signal transduction pathways. The genes from microarray expression study that are represented with red shades are upregulated, and green shades are downregulated, MMP16 was validated by qRT-PCR. The activity of ERK1/2 was assessed by MAPK activation dual detection assay flow cytometry. The red shade on functional assays denoted activation while green shade inhibition. Solid lines denoted direct interaction; interrupted lines denoted indirect interaction.
Figure Legend Snippet: Comprehensive VEGF signaling network of genes and proteins involved in cell migration and survival. Metformin-treated condition is compared to metformin-untreated condition under hyperglycemia-CoCl 2 for 12 h. The network was created by IPA software rendering VEGF signal transduction pathways. The genes from microarray expression study that are represented with red shades are upregulated, and green shades are downregulated, MMP16 was validated by qRT-PCR. The activity of ERK1/2 was assessed by MAPK activation dual detection assay flow cytometry. The red shade on functional assays denoted activation while green shade inhibition. Solid lines denoted direct interaction; interrupted lines denoted indirect interaction.

Techniques Used: Migration, Indirect Immunoperoxidase Assay, Software, Transduction, Microarray, Expressing, Quantitative RT-PCR, Activity Assay, Activation Assay, Detection Assay, Flow Cytometry, Cytometry, Functional Assay, Inhibition

19) Product Images from "Progression from High Insulin Resistance to Type 2 Diabetes Does Not Entail Additional Visceral Adipose Tissue Inflammation"

Article Title: Progression from High Insulin Resistance to Type 2 Diabetes Does Not Entail Additional Visceral Adipose Tissue Inflammation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048155

Intracellular inflammatory pathways in visceral adipose tissue from non obese, high IR-MO and T2D-MO individuals. ( A ) Expression and phosphorylation of JNK 1/2 isoforms: All obese patients, independently of high degree of insulin resistance and diabetes state, had significantly elevated levels of JNK 1 and 2 expression and phosphorylation versus lean group. ( B ) Expression and activation of ERK1/2 isoforms: All samples had similar levels of constitutive ERK 1/2 expression. VAT of lean subjects showed an undetectable ERK1/2 activation, in contrast to the high levels of ERK 1/2 activation seen in morbid obese patients (high IR-MO and T2D-MO). No significant differences were observed between both obese groups. ( C ) Expression and phosphorylation of STAT3: Both, phosphorylation and expression of STAT3 were significantly increased in VAT from MO individuals versus lean controls. Although there were not differences in the observed phosphorylation of STAT3 between high IR-MO and diabetic groups, elevated levels of expression were seen in diabetic individuals comparing with high IR-MO. ( D ) NF-κB pathway: There was a significant increase of IκB-α mRNA expression in VAT of MO compared to non obese subjects. Moreover, there were no significant differences in the high levels observed in both morbidly obese groups. EMSA technique revealed that NF-κB activation was significantly elevated in VAT of MO patients compared to the low levels seen in lean individuals. NF-κB activity levels were similar when the MO group was divided in high IR and T2D subgroup. Significant differences (Duncan; p
Figure Legend Snippet: Intracellular inflammatory pathways in visceral adipose tissue from non obese, high IR-MO and T2D-MO individuals. ( A ) Expression and phosphorylation of JNK 1/2 isoforms: All obese patients, independently of high degree of insulin resistance and diabetes state, had significantly elevated levels of JNK 1 and 2 expression and phosphorylation versus lean group. ( B ) Expression and activation of ERK1/2 isoforms: All samples had similar levels of constitutive ERK 1/2 expression. VAT of lean subjects showed an undetectable ERK1/2 activation, in contrast to the high levels of ERK 1/2 activation seen in morbid obese patients (high IR-MO and T2D-MO). No significant differences were observed between both obese groups. ( C ) Expression and phosphorylation of STAT3: Both, phosphorylation and expression of STAT3 were significantly increased in VAT from MO individuals versus lean controls. Although there were not differences in the observed phosphorylation of STAT3 between high IR-MO and diabetic groups, elevated levels of expression were seen in diabetic individuals comparing with high IR-MO. ( D ) NF-κB pathway: There was a significant increase of IκB-α mRNA expression in VAT of MO compared to non obese subjects. Moreover, there were no significant differences in the high levels observed in both morbidly obese groups. EMSA technique revealed that NF-κB activation was significantly elevated in VAT of MO patients compared to the low levels seen in lean individuals. NF-κB activity levels were similar when the MO group was divided in high IR and T2D subgroup. Significant differences (Duncan; p

Techniques Used: Expressing, Activation Assay, Activity Assay

20) Product Images from "Effects triggered by platinum nanoparticles on primary keratinocytes"

Article Title: Effects triggered by platinum nanoparticles on primary keratinocytes

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S49612

( A – D ) Activation of ERK, Akt, p38, and JNK by Western blot analysis. Normal human epidermal keratinocyte cells were cultured in 60 mm tissue-culture dishes at a density of 9.5 × 103 cells/cm 2 . The cells were exposed to 12.5 and 25 μg/mL PtNPs for 0.5, 4, and 24 hours. Untreated cells, harvested together at the 0.5-hour time point, served as a control. At the time of NP addition, the whole medium was exchanged in both treated and untreated cells. Tubulin was used as an internal control to monitor for equal loading. The data are representative of three independent experiments. Abbreviations: PtNPs, platinum nanoparticles; p-ERK, phosphorylated extracellular signal-regulated kinase; p-Akt, phosphorylated protein kinase B; p-JNK, phosphorylated c-Jun N-terminal kinase; Akt, protein kinase B.
Figure Legend Snippet: ( A – D ) Activation of ERK, Akt, p38, and JNK by Western blot analysis. Normal human epidermal keratinocyte cells were cultured in 60 mm tissue-culture dishes at a density of 9.5 × 103 cells/cm 2 . The cells were exposed to 12.5 and 25 μg/mL PtNPs for 0.5, 4, and 24 hours. Untreated cells, harvested together at the 0.5-hour time point, served as a control. At the time of NP addition, the whole medium was exchanged in both treated and untreated cells. Tubulin was used as an internal control to monitor for equal loading. The data are representative of three independent experiments. Abbreviations: PtNPs, platinum nanoparticles; p-ERK, phosphorylated extracellular signal-regulated kinase; p-Akt, phosphorylated protein kinase B; p-JNK, phosphorylated c-Jun N-terminal kinase; Akt, protein kinase B.

Techniques Used: Activation Assay, Western Blot, Cell Culture

21) Product Images from "Downstream-of-FGFR Is a Fibroblast Growth Factor-Specific Scaffolding Protein and Recruits Corkscrew upon Receptor Activation"

Article Title: Downstream-of-FGFR Is a Fibroblast Growth Factor-Specific Scaffolding Protein and Recruits Corkscrew upon Receptor Activation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.9.3769-3781.2004

(A) Activation of the MAPK pathway is important but not sufficient to direct cell migration. Activation of the MAPK pathway wasvisualized with anti-dpERK antibodies (subpanels a to d), the lumen of the tracheal system with the 2A12 antibody in red and terminal cells with the anti-DSRF antibody in green (subpanels e and f). Confocal projections are shown for embryos expressing the following transgenes under the control of the pannier Gal4 driver: UAS dof (subpanel a), UAS dof and UAS btl act (subpanel b), UAS dof600 ΔAR and UAS btl act (subpanel c) and UAS dof600Y515F and UAS btl act (subpanel d). (Subpanels e and f) Embryos expressing UAS dof600 ΔAR or UAS dof600Y515F in a dof −/− background under the control of the btl Gal4 driver are shown. (B) Summary table of the capacity of each transgene to activate dpERK in combination with an activated form of btl . The capacity of each dof transgene to rescue cell migration in dof −/− embryos is also shown in the second column.
Figure Legend Snippet: (A) Activation of the MAPK pathway is important but not sufficient to direct cell migration. Activation of the MAPK pathway wasvisualized with anti-dpERK antibodies (subpanels a to d), the lumen of the tracheal system with the 2A12 antibody in red and terminal cells with the anti-DSRF antibody in green (subpanels e and f). Confocal projections are shown for embryos expressing the following transgenes under the control of the pannier Gal4 driver: UAS dof (subpanel a), UAS dof and UAS btl act (subpanel b), UAS dof600 ΔAR and UAS btl act (subpanel c) and UAS dof600Y515F and UAS btl act (subpanel d). (Subpanels e and f) Embryos expressing UAS dof600 ΔAR or UAS dof600Y515F in a dof −/− background under the control of the btl Gal4 driver are shown. (B) Summary table of the capacity of each transgene to activate dpERK in combination with an activated form of btl . The capacity of each dof transgene to rescue cell migration in dof −/− embryos is also shown in the second column.

Techniques Used: Activation Assay, Migration, Expressing, Activated Clotting Time Assay

22) Product Images from "TRB3 Gene Silencing Alleviates Diabetic Cardiomyopathy in a Type 2 Diabetic Rat Model"

Article Title: TRB3 Gene Silencing Alleviates Diabetic Cardiomyopathy in a Type 2 Diabetic Rat Model

Journal: Diabetes

doi: 10.2337/db11-0549

TRB3 gene therapy improves cardiac function in diabetic (DM) rats. A : Relative mRNA expression and protein content of myocardial TRB3 in gene-silencing groups. B : Representative echocardiograms. C – G : Sequential evaluations of left ventricular end diastolic diameter (LVEDd) ( C ), FS ( D ), LVEF ( E ), E/A ( F ), and E′/A′ ( G ). H : Pressure curves of cardiac catheterization. I : Analysis of LVEDP with TRB3-siRNA silencing. Data are mean ± SEM; n = 7–10 per group. § P
Figure Legend Snippet: TRB3 gene therapy improves cardiac function in diabetic (DM) rats. A : Relative mRNA expression and protein content of myocardial TRB3 in gene-silencing groups. B : Representative echocardiograms. C – G : Sequential evaluations of left ventricular end diastolic diameter (LVEDd) ( C ), FS ( D ), LVEF ( E ), E/A ( F ), and E′/A′ ( G ). H : Pressure curves of cardiac catheterization. I : Analysis of LVEDP with TRB3-siRNA silencing. Data are mean ± SEM; n = 7–10 per group. § P

Techniques Used: Expressing

Diabetes increases collagen I (coll I) and collagen III (coll III) content, suppresses Akt phosphorylation, and partly activates TRB3/MAPK pathway. A : Representative immunohistochemical staining of collagen I and III (brown staining considered positive staining; scale bar: 50 μm). B : Representative Western blot of collagen I and III content. C : Western blot analysis of collagen I, collagen III, and collagen I-to-III ratio. D : Relative mRNA expression and protein content of myocardial TRB3. E : Western blot analysis of p-Akt/Akt, p-ERK/ERK, p-p38/p38, and p-JNK/JNK. Data are mean ± SEM. * P
Figure Legend Snippet: Diabetes increases collagen I (coll I) and collagen III (coll III) content, suppresses Akt phosphorylation, and partly activates TRB3/MAPK pathway. A : Representative immunohistochemical staining of collagen I and III (brown staining considered positive staining; scale bar: 50 μm). B : Representative Western blot of collagen I and III content. C : Western blot analysis of collagen I, collagen III, and collagen I-to-III ratio. D : Relative mRNA expression and protein content of myocardial TRB3. E : Western blot analysis of p-Akt/Akt, p-ERK/ERK, p-p38/p38, and p-JNK/JNK. Data are mean ± SEM. * P

Techniques Used: Immunohistochemistry, Staining, Western Blot, Expressing

TRB3 gene silencing decreases aberrant lipid accumulation and cardiac inflammation. A : Representative Oil Red O–stained myocardial sections (scale bar: 20 μm). B : Semiquantification of Oil Red O staining. Data are mean ± SEM of six independent observations in each group. C and D : Relative mRNA expression of myocardial TNF-α and IL-6. E : Immunohistochemical staining for myocardial TNF-α and IL-6 (brown staining considered positive staining; scale bar: 50 μm). F : Representative Western blot of myocardial TNF-α and IL-6. G and H : Western blot analyses of TNF-α ( G ) and IL-6 ( H ). Data are mean ± SEM. DM, diabetic rats. (A high-quality digital representation of this figure is available in the online issue.)
Figure Legend Snippet: TRB3 gene silencing decreases aberrant lipid accumulation and cardiac inflammation. A : Representative Oil Red O–stained myocardial sections (scale bar: 20 μm). B : Semiquantification of Oil Red O staining. Data are mean ± SEM of six independent observations in each group. C and D : Relative mRNA expression of myocardial TNF-α and IL-6. E : Immunohistochemical staining for myocardial TNF-α and IL-6 (brown staining considered positive staining; scale bar: 50 μm). F : Representative Western blot of myocardial TNF-α and IL-6. G and H : Western blot analyses of TNF-α ( G ) and IL-6 ( H ). Data are mean ± SEM. DM, diabetic rats. (A high-quality digital representation of this figure is available in the online issue.)

Techniques Used: Staining, Expressing, Immunohistochemistry, Western Blot

TRB3 gene silencing decreases collagen expression, restores the phosphorylation of p-Akt, and partly reverses MAPK activation. A : Representative immunohistochemical staining showing collagen I and III (scale bar: 50 μm). B : Representative Western blot of collagen I and III. C : Western blot analyses of collagen I and III content and collagen I-to-III ratio. D : Western blot analysis of p-Akt/Akt, p-ERK/ERK, p-p38/p38MAPK, and p-JNK/JNK. Data are mean ± SEM. DM, diabetic rats. (A high-quality digital representation of this figure is available in the online issue.)
Figure Legend Snippet: TRB3 gene silencing decreases collagen expression, restores the phosphorylation of p-Akt, and partly reverses MAPK activation. A : Representative immunohistochemical staining showing collagen I and III (scale bar: 50 μm). B : Representative Western blot of collagen I and III. C : Western blot analyses of collagen I and III content and collagen I-to-III ratio. D : Western blot analysis of p-Akt/Akt, p-ERK/ERK, p-p38/p38MAPK, and p-JNK/JNK. Data are mean ± SEM. DM, diabetic rats. (A high-quality digital representation of this figure is available in the online issue.)

Techniques Used: Expressing, Activation Assay, Immunohistochemistry, Staining, Western Blot

TRB3 gene therapy decreases myocardial hypertrophy and fibrosis in HF and diabetic (DM) groups. A1 : Heart size (scale bar: 2 mm). A2 : Representative cross sections of hearts at the papillary muscle level (scale bar: 2 mm). A3 : Longitudinal sections of LV stained with hematoxylin and eosin (H E) (scale bar: 50 μm). A4 : Transverse section of LV stained with H E (scale bar: 50 μm). B – D : Quantitative data for heart weight to body weight (HW-to-BW) ratio, myocyte size, and relative brain natriuretic protein (BNP) mRNA expression. E1–6 : Representative Masson trichrome staining and Picrosirius red staining. F – H : Quantitative analyses of CVF, collagen content, and PVCA/LA. Data are mean ± SEM; n = 7–10 per group. (A high-quality digital representation of this figure is available in the online issue.)
Figure Legend Snippet: TRB3 gene therapy decreases myocardial hypertrophy and fibrosis in HF and diabetic (DM) groups. A1 : Heart size (scale bar: 2 mm). A2 : Representative cross sections of hearts at the papillary muscle level (scale bar: 2 mm). A3 : Longitudinal sections of LV stained with hematoxylin and eosin (H E) (scale bar: 50 μm). A4 : Transverse section of LV stained with H E (scale bar: 50 μm). B – D : Quantitative data for heart weight to body weight (HW-to-BW) ratio, myocyte size, and relative brain natriuretic protein (BNP) mRNA expression. E1–6 : Representative Masson trichrome staining and Picrosirius red staining. F – H : Quantitative analyses of CVF, collagen content, and PVCA/LA. Data are mean ± SEM; n = 7–10 per group. (A high-quality digital representation of this figure is available in the online issue.)

Techniques Used: Staining, Expressing

23) Product Images from "Neuroprotective Effects of a Smoothened Receptor Agonist against Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats"

Article Title: Neuroprotective Effects of a Smoothened Receptor Agonist against Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2016.00306

Effects of treatment with PUR on Bax, Bcl-2 in mRNA and protein levels. (A) The relative expression levels of Bax and Bcl-2 mRNA in the PFC were analyzed by RT-PCR. The densities of the protein bands were analyzed and normalized to β-actin, n = 3. (B) Representative western blots showing levels of Bax and Bcl-2 in the PFC, Bar graphs showing quantification of the protein levels of Bax and Bcl-2, n = 3. Both the determination of the two signs in mRNA and protein levels were obtained from three separate experiments. Values represent the mean ± SD. *** p
Figure Legend Snippet: Effects of treatment with PUR on Bax, Bcl-2 in mRNA and protein levels. (A) The relative expression levels of Bax and Bcl-2 mRNA in the PFC were analyzed by RT-PCR. The densities of the protein bands were analyzed and normalized to β-actin, n = 3. (B) Representative western blots showing levels of Bax and Bcl-2 in the PFC, Bar graphs showing quantification of the protein levels of Bax and Bcl-2, n = 3. Both the determination of the two signs in mRNA and protein levels were obtained from three separate experiments. Values represent the mean ± SD. *** p

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

Effects of PUR on Shh pathway . The quantification of Shh, Gli-1, and Ptch was measured by RT-PCR (A) and western blot (B) 48 h post-SAH. Each value was normalized to β-actin. Bar graphs showing quantification of mRNA and protein levels of Shh, Gli-1, and Ptch were determined by Image-Pro Plus 6.0, n = 3. Values represent the mean ± SD. ** p
Figure Legend Snippet: Effects of PUR on Shh pathway . The quantification of Shh, Gli-1, and Ptch was measured by RT-PCR (A) and western blot (B) 48 h post-SAH. Each value was normalized to β-actin. Bar graphs showing quantification of mRNA and protein levels of Shh, Gli-1, and Ptch were determined by Image-Pro Plus 6.0, n = 3. Values represent the mean ± SD. ** p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot

24) Product Images from "The Epidermal Growth Factor-like Domain of CD93 Is a Potent Angiogenic Factor"

Article Title: The Epidermal Growth Factor-like Domain of CD93 Is a Potent Angiogenic Factor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0051647

rCD93D23 induces tube formation through the activation of Akt and ERK in HUVECs on Matrigel in vitro . (A) rCD93D23 induced tube formation in a dose-dependent manner. (B) The tube formation induced by boiled rCD93D23, and rCD93D23 in the presence of polyclonal anti-CD93D23 IgG (10 µg/ml), U0126 (10 µM), or LY294002 (10 µM). Each value represents the mean ± SD (n = 3), and similar results were obtained in three independent experiments. **, p
Figure Legend Snippet: rCD93D23 induces tube formation through the activation of Akt and ERK in HUVECs on Matrigel in vitro . (A) rCD93D23 induced tube formation in a dose-dependent manner. (B) The tube formation induced by boiled rCD93D23, and rCD93D23 in the presence of polyclonal anti-CD93D23 IgG (10 µg/ml), U0126 (10 µM), or LY294002 (10 µM). Each value represents the mean ± SD (n = 3), and similar results were obtained in three independent experiments. **, p

Techniques Used: Activation Assay, In Vitro

rCD93D23 induces HUVEC migration through the activation of Akt and ERK. (A) rCD93D23 dose-dependently induced FAK, ERK1/2, Akt, and eNOS phosphorylation. HUVECs were treated with different concentrations of rCD93D23 or VEGF (10 ng/ml) for 30 min. (B) rCD93D23 time-dependently induced FAK, ERK1/2, Akt, and eNOS phosphorylation in HUVECs. HUVECs were stimulated with 50 ng/ml of rCD93D23 for various time periods as indicated. The similar results were obtained in three independent experiments. *, p
Figure Legend Snippet: rCD93D23 induces HUVEC migration through the activation of Akt and ERK. (A) rCD93D23 dose-dependently induced FAK, ERK1/2, Akt, and eNOS phosphorylation. HUVECs were treated with different concentrations of rCD93D23 or VEGF (10 ng/ml) for 30 min. (B) rCD93D23 time-dependently induced FAK, ERK1/2, Akt, and eNOS phosphorylation in HUVECs. HUVECs were stimulated with 50 ng/ml of rCD93D23 for various time periods as indicated. The similar results were obtained in three independent experiments. *, p

Techniques Used: Migration, Activation Assay

25) Product Images from "Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways"

Article Title: Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

doi: 10.4196/kjpp.2015.19.3.219

Neuroprotective effects of the blockade of PI3K/Akt and NADPH oxidase against microglia-mediated neuronal cell death. Neuroprotective effects of the blockade of PI3K/Akt and NADPH oxidase were investigated against microglia-mediated neuronal cell death in microglial conditioned media system (A) and in neuronmicroglia co-culture system (B). Wortmannin (100 nM), LY294002 (25 µM) and apocynin (500 µM) were pretreated in both experimental systems and then, neuronal cell deaths were evaluated as shown in Fig. 2 (See 'Materials and methods' for the detailed description). Values are the mean±S.E.M. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. ### p
Figure Legend Snippet: Neuroprotective effects of the blockade of PI3K/Akt and NADPH oxidase against microglia-mediated neuronal cell death. Neuroprotective effects of the blockade of PI3K/Akt and NADPH oxidase were investigated against microglia-mediated neuronal cell death in microglial conditioned media system (A) and in neuronmicroglia co-culture system (B). Wortmannin (100 nM), LY294002 (25 µM) and apocynin (500 µM) were pretreated in both experimental systems and then, neuronal cell deaths were evaluated as shown in Fig. 2 (See 'Materials and methods' for the detailed description). Values are the mean±S.E.M. of four samples in one independent experiment. The data were replicated in three repeated independent experiments. ### p

Techniques Used: Co-Culture Assay

26) Product Images from "Critical Role for Transcription Factor C/EBP-β in Regulating the Expression of Death-Associated Protein Kinase 1 "

Article Title: Critical Role for Transcription Factor C/EBP-β in Regulating the Expression of Death-Associated Protein Kinase 1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00784-07

Effect of IFN-γ on the binding of C/EBP-β to the distal CBS and proximal CRE/ATF sites of the dapk1 promoter in MEFs. ChIP assays were performed using a commercially available kit (Upstate Biotech, Inc.). (A and C) Typical PCR patterns obtained in ChIP assays with distal CBS-specific (A) and proximal CRE/ATF-specific (C) primers (see Table S5 in the supplemental material). For input control reactions, one-fifth of the soluble chromatin used for the ChIP analysis was employed. Twenty-six cycles of PCR were performed in each case. NR IgG, normal rabbit IgG. C/EBP and IgG were used at 10 μg each/reaction. (B and D) Real-time PCR analysis of dapk1 promoter fragments recovered in ChIP assays performed with the indicated antibodies using chromatin prepared from wild-type and cebpb −/− MEFs. Each bar represents the mean abundance of dapk1 promoter fragments ± SE of six separate reactions from two independent experiments. (E) Wild-type MEFs were transfected with constructs carrying only the distal CBS or the proximal CRE/ATF (shown at the bottom of D) for 36 h. After stimulation with IFN-γ, chromatin was prepared and used for ChIP assays. For the distal CBS construct, primers 180mDAPK-R and RV3 were used for PCR. For the proximal CRE, primers 200mDAPK-F and GL2 were used for PCR. The rest of the controls for this experiment were essentially similar to those described above (A). (F) ChIP analysis of cells transfected with the single- and double-mutant constructs shown at bottom of panel C. The plasmid-specific primers RV3 and GL2 were used for PCR analysis of the ChIP products. (G) Comparison of the sequences of CBS and CRE/ATF elements of the dapk1 promoter. Core sequences are shown in boldface, italicized type. Vertical lines show the homologous nucleotides.
Figure Legend Snippet: Effect of IFN-γ on the binding of C/EBP-β to the distal CBS and proximal CRE/ATF sites of the dapk1 promoter in MEFs. ChIP assays were performed using a commercially available kit (Upstate Biotech, Inc.). (A and C) Typical PCR patterns obtained in ChIP assays with distal CBS-specific (A) and proximal CRE/ATF-specific (C) primers (see Table S5 in the supplemental material). For input control reactions, one-fifth of the soluble chromatin used for the ChIP analysis was employed. Twenty-six cycles of PCR were performed in each case. NR IgG, normal rabbit IgG. C/EBP and IgG were used at 10 μg each/reaction. (B and D) Real-time PCR analysis of dapk1 promoter fragments recovered in ChIP assays performed with the indicated antibodies using chromatin prepared from wild-type and cebpb −/− MEFs. Each bar represents the mean abundance of dapk1 promoter fragments ± SE of six separate reactions from two independent experiments. (E) Wild-type MEFs were transfected with constructs carrying only the distal CBS or the proximal CRE/ATF (shown at the bottom of D) for 36 h. After stimulation with IFN-γ, chromatin was prepared and used for ChIP assays. For the distal CBS construct, primers 180mDAPK-R and RV3 were used for PCR. For the proximal CRE, primers 200mDAPK-F and GL2 were used for PCR. The rest of the controls for this experiment were essentially similar to those described above (A). (F) ChIP analysis of cells transfected with the single- and double-mutant constructs shown at bottom of panel C. The plasmid-specific primers RV3 and GL2 were used for PCR analysis of the ChIP products. (G) Comparison of the sequences of CBS and CRE/ATF elements of the dapk1 promoter. Core sequences are shown in boldface, italicized type. Vertical lines show the homologous nucleotides.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection, Construct, Mutagenesis, Plasmid Preparation

ERK-dependent regulation and phosphorylation of C/EBP-β in response to IFN-γ. (A) Activation of ERKs in wild-type and erk1 −/− MEFs. Cells were stimulated with IFN-γ for 1 h, and lysates (30 μg) were subjected to Western blot (WB) analysis with phospho-ERK-specific (activated form) and native ERK2-specific antibodies. (B) ERK1 and ERK2 are critical for the IFN-γ-induced expression of dapk1 mRNA. Real-time PCR analysis of the dapk1 transcript is shown. The middle part of this graph shows the effect of ERK2 knockdown in erk1 −/− MEFs on DAPK1 expression. The last four bars show the results of a rescue experiment. Following the knockdown of erk2 expression in erk1 −/− MEFs with shRNAs, cells were transfected with an expression vector coding for murine ERK1. shE2, erk2 -specific shRNA; SC, scrambled shRNA; EV, empty vector. (C) Effects of lentivirus-encoded erk2 shRNAs on ERK2 expression in erk1 −/− MEFs. Western blot analysis of the cell lysates (45 μg) is shown. (D) Cells infected with lentiviral vector erk2 -specific shRNAs (see panel C) were electroporated with empty vector (pCDNA3.1) or the same vector coding for ERK1. Western blot analysis of the lysates was performed with the indicated antibodies. (E) Phosphorylation of recombinant C/EBP-β protein by ERK1/2. Wild-type MEFs were stimulated with IFN-γ for 2 h, and ERK1 and ERK2 were immunoprecipitated using protein G/protein A agarose beads (Santa Cruz Biotech). The bead-bound IP products were washed twice with the reaction buffer prior to incubation with recombinant C/EBP-β (2 μg) in the presence of [ 32 P]ATP. The beads were spun down, and supernatants were collected, separated by SDS-PAGE, and transferred onto a nylon membrane prior to autoradiography. A typical profile is shown. These reactions were also probed with ERK2-, ppERK1-, and C/EBP-β-specific antibodies. (F) Quantification of ERK1/2-induced C/EBP-β phosphorylation. Lysates were immunoprecipitated with ERK1/2 antibodies (lanes 1 and 2), normal rabbit antibody (lanes 3 and 4), and no antibody (lanes 5 and 6), and in vitro phosphorylation was performed as described below (G). The reaction products were separated by SDS-PAGE and transferred onto a nylon membrane prior to phosphorimaging. Radioactive signals (arbitrary units) in the C/EBP-β band were quantified and presented. Each bar shows the mean ± SE ( n = 3). (G) In vitro phosphorylation of C/EBP-β was performed as described above (E), except that cold ATP was used for phosphorylation. Following this, the reaction products were subjected to a Western blot analysis with native and C/EBP-β-phospho-T 189 -specific antibodies.
Figure Legend Snippet: ERK-dependent regulation and phosphorylation of C/EBP-β in response to IFN-γ. (A) Activation of ERKs in wild-type and erk1 −/− MEFs. Cells were stimulated with IFN-γ for 1 h, and lysates (30 μg) were subjected to Western blot (WB) analysis with phospho-ERK-specific (activated form) and native ERK2-specific antibodies. (B) ERK1 and ERK2 are critical for the IFN-γ-induced expression of dapk1 mRNA. Real-time PCR analysis of the dapk1 transcript is shown. The middle part of this graph shows the effect of ERK2 knockdown in erk1 −/− MEFs on DAPK1 expression. The last four bars show the results of a rescue experiment. Following the knockdown of erk2 expression in erk1 −/− MEFs with shRNAs, cells were transfected with an expression vector coding for murine ERK1. shE2, erk2 -specific shRNA; SC, scrambled shRNA; EV, empty vector. (C) Effects of lentivirus-encoded erk2 shRNAs on ERK2 expression in erk1 −/− MEFs. Western blot analysis of the cell lysates (45 μg) is shown. (D) Cells infected with lentiviral vector erk2 -specific shRNAs (see panel C) were electroporated with empty vector (pCDNA3.1) or the same vector coding for ERK1. Western blot analysis of the lysates was performed with the indicated antibodies. (E) Phosphorylation of recombinant C/EBP-β protein by ERK1/2. Wild-type MEFs were stimulated with IFN-γ for 2 h, and ERK1 and ERK2 were immunoprecipitated using protein G/protein A agarose beads (Santa Cruz Biotech). The bead-bound IP products were washed twice with the reaction buffer prior to incubation with recombinant C/EBP-β (2 μg) in the presence of [ 32 P]ATP. The beads were spun down, and supernatants were collected, separated by SDS-PAGE, and transferred onto a nylon membrane prior to autoradiography. A typical profile is shown. These reactions were also probed with ERK2-, ppERK1-, and C/EBP-β-specific antibodies. (F) Quantification of ERK1/2-induced C/EBP-β phosphorylation. Lysates were immunoprecipitated with ERK1/2 antibodies (lanes 1 and 2), normal rabbit antibody (lanes 3 and 4), and no antibody (lanes 5 and 6), and in vitro phosphorylation was performed as described below (G). The reaction products were separated by SDS-PAGE and transferred onto a nylon membrane prior to phosphorimaging. Radioactive signals (arbitrary units) in the C/EBP-β band were quantified and presented. Each bar shows the mean ± SE ( n = 3). (G) In vitro phosphorylation of C/EBP-β was performed as described above (E), except that cold ATP was used for phosphorylation. Following this, the reaction products were subjected to a Western blot analysis with native and C/EBP-β-phospho-T 189 -specific antibodies.

Techniques Used: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Infection, Recombinant, Immunoprecipitation, Incubation, SDS Page, Autoradiography, In Vitro

The T 189 residue within RD2 of C/EBP-β is required for the IFN-γ-inducible expression of DAPK1. (A) Mutations introduced into C/EBP-β cDNA. TAD, transcription activation domain; Basic, domain rich in basic amino acids; LZ, leucine zipper domain. The mutated amino acids are indicated in each case. (B and C) Effect of cebpb mutations on the IFN-induced expression of dapk1. cebpb −/− MEFs were electroporated with the indicated mutants along with m dapk 1.2K-Luc. A portion of the transfected cells was used for the luciferase assay, and the remainder was used for real-time PCR analysis of the dapk1 transcript. (D) Expression of mutant and wild-type (Wt) versions of C/EBP-β in the lysates of transfected cells. Western blot (WB) analysis with indicated antibodies was conducted using 100 μg of total protein. (E) Quantitative ChIP assay for the recruitment of C/EBP-β to the CRE/ATF site. IFN-γ treatment was done for 12 h.
Figure Legend Snippet: The T 189 residue within RD2 of C/EBP-β is required for the IFN-γ-inducible expression of DAPK1. (A) Mutations introduced into C/EBP-β cDNA. TAD, transcription activation domain; Basic, domain rich in basic amino acids; LZ, leucine zipper domain. The mutated amino acids are indicated in each case. (B and C) Effect of cebpb mutations on the IFN-induced expression of dapk1. cebpb −/− MEFs were electroporated with the indicated mutants along with m dapk 1.2K-Luc. A portion of the transfected cells was used for the luciferase assay, and the remainder was used for real-time PCR analysis of the dapk1 transcript. (D) Expression of mutant and wild-type (Wt) versions of C/EBP-β in the lysates of transfected cells. Western blot (WB) analysis with indicated antibodies was conducted using 100 μg of total protein. (E) Quantitative ChIP assay for the recruitment of C/EBP-β to the CRE/ATF site. IFN-γ treatment was done for 12 h.

Techniques Used: Expressing, Activation Assay, Transfection, Luciferase, Real-time Polymerase Chain Reaction, Mutagenesis, Western Blot, Chromatin Immunoprecipitation

Restoration of dapk1 expression reestablishes IFN-γ-induced apoptosis in cebpb −/− BM cells. (A) Cells were electroporated with 4 μg of an empty pcDNA3.1 vector or the same vector expressing either the wild-type or the kinase-deficient (KD) mutant (K42A) of DAPK1 along with the pEGFP expression vector (2 μg) for tracking the transfected cells. The cells were then treated with IFN-γ (500 U/ml) for 24 h and stained with TRITC-annexin V (red fluorescence). Cells were subjected to FACS analysis. Green-positive (GFP-positive) and green/red-positive (GFP-positive/TRITC-positive) cells were estimated. The double-positive cells were estimated as a percentage of total GFP-positive cells to determine the magnitude of cell death. (B) Western blot (WB) analysis of the DAPK1 expression. A portion of transfected and IFN-treated cells was lysed, and the expression levels of DAPK1 and GFP were monitored with specific antibodies.
Figure Legend Snippet: Restoration of dapk1 expression reestablishes IFN-γ-induced apoptosis in cebpb −/− BM cells. (A) Cells were electroporated with 4 μg of an empty pcDNA3.1 vector or the same vector expressing either the wild-type or the kinase-deficient (KD) mutant (K42A) of DAPK1 along with the pEGFP expression vector (2 μg) for tracking the transfected cells. The cells were then treated with IFN-γ (500 U/ml) for 24 h and stained with TRITC-annexin V (red fluorescence). Cells were subjected to FACS analysis. Green-positive (GFP-positive) and green/red-positive (GFP-positive/TRITC-positive) cells were estimated. The double-positive cells were estimated as a percentage of total GFP-positive cells to determine the magnitude of cell death. (B) Western blot (WB) analysis of the DAPK1 expression. A portion of transfected and IFN-treated cells was lysed, and the expression levels of DAPK1 and GFP were monitored with specific antibodies.

Techniques Used: Expressing, Plasmid Preparation, Mutagenesis, Transfection, Staining, Fluorescence, FACS, Western Blot

Basal and IFN-γ-induced expression of dapk1 is C/EBP-β dependent. (A) cebpb +/+ and cebpb −/− cells were stimulated with IFN-γ for various hours, and poly(A) + RNA was prepared. About 3 μg of RNA was used for Northern blot (NB) analysis with the indicated gene probes labeled with 32 P. The blots were washed and exposed to X-ray films to detect the bands. Blot regions corresponding to the specific bands are shown. (B) Activation of STAT1 via phosphorylation at tyrosine 701 by IFN-γ treatment. The upper and lower bands correspond to the STAT1α and STAT1β proteins, respectively. (C) Expression of endogenous dapk1 mRNA and protein in cebpb +/+ and cebpb −/− MEFs. RT-PCR with gene-specific primers was used to detect the specific transcripts indicated on the right. RT-PCR for irf1 and dapk1 was performed with RNA isolated after 6 and 12 h of IFN treatment, respectively. Western blot (WB) analysis was performed with specific antibodies after separating 85 μg of total protein by SDS-PAGE and Western transfer. (D) Nuclear runoff transcription. Nuclei were isolated after stimulating the BM cells with IFN-γ for 8 h and incubated with 32 P-lableled UTP for 45 min. The labeled RNAs were extracted and hybridized to specific DNAs affixed on a nylon filter. pcDNA3.1 and gapdh were used as negative and positive controls, respectively, in this experiment. (E) RT-PCR analysis of the expression of dapk1 , irf1 , and gapdh transcripts using RNA extracted form IFN-γ-treated cebpd +/+ and cebpd −/− MEFs. (F and G) Effect of shRNA-mediated knocking down of cebpa and cebpd on the IFN-induced expression of dapk 1 . Wild-type BM cells were infected with lentiviral vectors coding for cebpa - and cebpd -specific shRNAs or the control empty vector (pLKO-1). Real-time PCR was carried out using dapk1 -specific primers. (G) Western blot analysis of cell extracts (80 μg from each sample). (H and K) Effect of cebpb -specific shRNAs on the expression of endogenous C/EBP-β in MEFs (H) and hTERT-HME1 (K) cells. Lentiviral vector (pLKO-1)-encoded species-specific shRNAs against C/EBP-β transcript were used for knocking down the expression of endogenous C/EBP-β. (I and L) Real-time PCR analysis of IFN-induced expression of dapk1 and irf1 transcripts in MEFs (I) and hTERT-HME1 (L) cells. (J and M) Western blot analyses of the DAPK1 and actin proteins in MEFs (J) and hTERT-HME1 (M) cells.
Figure Legend Snippet: Basal and IFN-γ-induced expression of dapk1 is C/EBP-β dependent. (A) cebpb +/+ and cebpb −/− cells were stimulated with IFN-γ for various hours, and poly(A) + RNA was prepared. About 3 μg of RNA was used for Northern blot (NB) analysis with the indicated gene probes labeled with 32 P. The blots were washed and exposed to X-ray films to detect the bands. Blot regions corresponding to the specific bands are shown. (B) Activation of STAT1 via phosphorylation at tyrosine 701 by IFN-γ treatment. The upper and lower bands correspond to the STAT1α and STAT1β proteins, respectively. (C) Expression of endogenous dapk1 mRNA and protein in cebpb +/+ and cebpb −/− MEFs. RT-PCR with gene-specific primers was used to detect the specific transcripts indicated on the right. RT-PCR for irf1 and dapk1 was performed with RNA isolated after 6 and 12 h of IFN treatment, respectively. Western blot (WB) analysis was performed with specific antibodies after separating 85 μg of total protein by SDS-PAGE and Western transfer. (D) Nuclear runoff transcription. Nuclei were isolated after stimulating the BM cells with IFN-γ for 8 h and incubated with 32 P-lableled UTP for 45 min. The labeled RNAs were extracted and hybridized to specific DNAs affixed on a nylon filter. pcDNA3.1 and gapdh were used as negative and positive controls, respectively, in this experiment. (E) RT-PCR analysis of the expression of dapk1 , irf1 , and gapdh transcripts using RNA extracted form IFN-γ-treated cebpd +/+ and cebpd −/− MEFs. (F and G) Effect of shRNA-mediated knocking down of cebpa and cebpd on the IFN-induced expression of dapk 1 . Wild-type BM cells were infected with lentiviral vectors coding for cebpa - and cebpd -specific shRNAs or the control empty vector (pLKO-1). Real-time PCR was carried out using dapk1 -specific primers. (G) Western blot analysis of cell extracts (80 μg from each sample). (H and K) Effect of cebpb -specific shRNAs on the expression of endogenous C/EBP-β in MEFs (H) and hTERT-HME1 (K) cells. Lentiviral vector (pLKO-1)-encoded species-specific shRNAs against C/EBP-β transcript were used for knocking down the expression of endogenous C/EBP-β. (I and L) Real-time PCR analysis of IFN-induced expression of dapk1 and irf1 transcripts in MEFs (I) and hTERT-HME1 (L) cells. (J and M) Western blot analyses of the DAPK1 and actin proteins in MEFs (J) and hTERT-HME1 (M) cells.

Techniques Used: Expressing, Northern Blot, Labeling, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot, SDS Page, Incubation, shRNA, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction

27) Product Images from "TRPA1 is required for TGF-β signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma"

Article Title: TRPA1 is required for TGF-β signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma

Journal: Laboratory investigation; a journal of technical methods and pathology

doi: 10.1038/labinvest.2014.85

Western blot analysis of Smad3, p38 MAPK, ERK, and JNK signaling upon exposure to TGF- β 1. Adding transforming growth factor- β 1 (TGF- β 1) to the culture medium upregulated the phosphorylation of Smad3 in 15 min, and even at 300 min phospho-Smad3 was detected in cultured ocular fibroblasts. Similarly, adding TGF- β 1 to the culture medium upregulated the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK) in 30 min, and phosphor-p38 MAPK and phospho-ERK were maintained at 300 min. Phosphorylation of c-Jun N-terminal kinase (JNK) was seen in 15 min, and even at 300 min phosphor-JNK was detected in cultured ocular fibroblasts. A loss of TRPA1 in the cells blocked the TGF- β 1-induced phosphorylation of Smad3, p38 MAPK, ERK, and JNK.
Figure Legend Snippet: Western blot analysis of Smad3, p38 MAPK, ERK, and JNK signaling upon exposure to TGF- β 1. Adding transforming growth factor- β 1 (TGF- β 1) to the culture medium upregulated the phosphorylation of Smad3 in 15 min, and even at 300 min phospho-Smad3 was detected in cultured ocular fibroblasts. Similarly, adding TGF- β 1 to the culture medium upregulated the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK) in 30 min, and phosphor-p38 MAPK and phospho-ERK were maintained at 300 min. Phosphorylation of c-Jun N-terminal kinase (JNK) was seen in 15 min, and even at 300 min phosphor-JNK was detected in cultured ocular fibroblasts. A loss of TRPA1 in the cells blocked the TGF- β 1-induced phosphorylation of Smad3, p38 MAPK, ERK, and JNK.

Techniques Used: Western Blot, Cell Culture

28) Product Images from "Breviscapine ameliorates CCl4-induced liver injury in mice through inhibiting inflammatory apoptotic response and ROS generation"

Article Title: Breviscapine ameliorates CCl4-induced liver injury in mice through inhibiting inflammatory apoptotic response and ROS generation

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3651

Breviscapine suppresses the inflammatory response in L02 cells stimulated with LPS in vitro . (A) The normal liver cell lines of L02, BRL-3A and AML-12 were treated with breviscapine at different concentrations (0, 5, 10, 20 and 40 µ M) for various durations ranging from 0-72 h. The cell viability was measured by a cell counting kit-8 assay to evaluate the cytotoxicity of breviscapine to liver cells. In addition, L02 cells were exposed to 100 ng/ml LPS for 24 h in the absence or presence of breviscapine (20 or 40 µ M). (B) The pro-inflammatory cytokines TNF-α, IL-6, IL-1β and IL-18, and the chemokine MCP-1 were assessed by reverse transcription-quantitative polymerase chain reaction analysis. (C) Western blot analysis was used to evaluate TLR4 and MyD88 levels in L02 cells after various treatments. (D) IKKα, IκBα and NF-κB (p65) phosphorylation were examined by immunoblotting analysis. Representative western blot images and quantified protein levels are provided. Values are expressed as the mean ± standard error of the mean (n=8). * P
Figure Legend Snippet: Breviscapine suppresses the inflammatory response in L02 cells stimulated with LPS in vitro . (A) The normal liver cell lines of L02, BRL-3A and AML-12 were treated with breviscapine at different concentrations (0, 5, 10, 20 and 40 µ M) for various durations ranging from 0-72 h. The cell viability was measured by a cell counting kit-8 assay to evaluate the cytotoxicity of breviscapine to liver cells. In addition, L02 cells were exposed to 100 ng/ml LPS for 24 h in the absence or presence of breviscapine (20 or 40 µ M). (B) The pro-inflammatory cytokines TNF-α, IL-6, IL-1β and IL-18, and the chemokine MCP-1 were assessed by reverse transcription-quantitative polymerase chain reaction analysis. (C) Western blot analysis was used to evaluate TLR4 and MyD88 levels in L02 cells after various treatments. (D) IKKα, IκBα and NF-κB (p65) phosphorylation were examined by immunoblotting analysis. Representative western blot images and quantified protein levels are provided. Values are expressed as the mean ± standard error of the mean (n=8). * P

Techniques Used: In Vitro, Cell Counting, Real-time Polymerase Chain Reaction, Western Blot

Breviscapine downregulates LPS-induced oxidative stress in L02 cells. (A) A 2′,7′-dichlorofluoresceindiacetate assay was performed to measure the generation of reactive oxygen species in L02 cells treated with LPS with or without breviscapine treatment (20 or 40 µ M). Scale bar, 50 µ m. (B) SOD1, NQO-1, HO-1 and Nrf2, as well as (C) p-p38, p-ERK1/2 and p-JNK protein levels were measured using western blot analysis. The quantified results are provided in the bar graphs. Values are expressed as the mean ± standard error of the mean (n=10). *** P
Figure Legend Snippet: Breviscapine downregulates LPS-induced oxidative stress in L02 cells. (A) A 2′,7′-dichlorofluoresceindiacetate assay was performed to measure the generation of reactive oxygen species in L02 cells treated with LPS with or without breviscapine treatment (20 or 40 µ M). Scale bar, 50 µ m. (B) SOD1, NQO-1, HO-1 and Nrf2, as well as (C) p-p38, p-ERK1/2 and p-JNK protein levels were measured using western blot analysis. The quantified results are provided in the bar graphs. Values are expressed as the mean ± standard error of the mean (n=10). *** P

Techniques Used: Western Blot

Breviscapine ameliorates CCl 4 -induced liver injury by reducing pro-inflammatory cytokine secretion. (A) Serum pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and chemokine of MCP-1 were determined using ELISA kits. (B) Liver pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and chemokine MCP-1 were determined using commercial ELISA kits. Values are expressed as the mean ± standard error of the mean (n=10). ** P
Figure Legend Snippet: Breviscapine ameliorates CCl 4 -induced liver injury by reducing pro-inflammatory cytokine secretion. (A) Serum pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and chemokine of MCP-1 were determined using ELISA kits. (B) Liver pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and chemokine MCP-1 were determined using commercial ELISA kits. Values are expressed as the mean ± standard error of the mean (n=10). ** P

Techniques Used: Enzyme-linked Immunosorbent Assay

Breviscapine attenuates liver injury by inhibiting apoptosis in CCl 4 -induced mice. (A) Immunohistochemical assays were performed to evaluate Bax levels in the liver sections of mice subjected to different treatments. Scale bar, 100 µ m. (B) Bax and Bcl-2 protein levels were evaluated by western blot analysis. Quantified levels of Bax and Bcl-2 are displayed, and the ratio of Bax/Bcl-2 was exhibited. (C) Active caspase-3 and PARP-1 protein abundance was determined by western blot analysis. Representative blot images of proteins are displayed, accompanied with the quantified levels. Values are expressed as the mean ± standard error of the mean (n=10). *** P
Figure Legend Snippet: Breviscapine attenuates liver injury by inhibiting apoptosis in CCl 4 -induced mice. (A) Immunohistochemical assays were performed to evaluate Bax levels in the liver sections of mice subjected to different treatments. Scale bar, 100 µ m. (B) Bax and Bcl-2 protein levels were evaluated by western blot analysis. Quantified levels of Bax and Bcl-2 are displayed, and the ratio of Bax/Bcl-2 was exhibited. (C) Active caspase-3 and PARP-1 protein abundance was determined by western blot analysis. Representative blot images of proteins are displayed, accompanied with the quantified levels. Values are expressed as the mean ± standard error of the mean (n=10). *** P

Techniques Used: Mouse Assay, Immunohistochemistry, Western Blot

Breviscapine reduces apoptosis the liver of mice treated with CCl 4 . (A) TUNEL analysis was used to assess apoptosis in CCl 4 -induced livers in mice after Breviscapine administration. Scale bar, 100 µ m. (B) Apaf-1 positive cells were calculated in the liver sections of mice. Scale bar, 100 µ m. Values are expressed as the mean ± standard error of the mean (n=10). *** P
Figure Legend Snippet: Breviscapine reduces apoptosis the liver of mice treated with CCl 4 . (A) TUNEL analysis was used to assess apoptosis in CCl 4 -induced livers in mice after Breviscapine administration. Scale bar, 100 µ m. (B) Apaf-1 positive cells were calculated in the liver sections of mice. Scale bar, 100 µ m. Values are expressed as the mean ± standard error of the mean (n=10). *** P

Techniques Used: Mouse Assay, TUNEL Assay

Breviscapine attenuates liver injury in mice induced by CCl 4 . (A) ALT, (B) AST and (C) Albumin levels in the serum of mice were examined to assess the effect of breviscapine in regulating liver injury induced by CCl 4 . (D) Liver hydroxyproline levels were assessed. Values are expressed as the mean ± standard error of the mean (n=10). *** P
Figure Legend Snippet: Breviscapine attenuates liver injury in mice induced by CCl 4 . (A) ALT, (B) AST and (C) Albumin levels in the serum of mice were examined to assess the effect of breviscapine in regulating liver injury induced by CCl 4 . (D) Liver hydroxyproline levels were assessed. Values are expressed as the mean ± standard error of the mean (n=10). *** P

Techniques Used: Mouse Assay, AST Assay

Breviscapine reduces oxidative stress in the livers of mice induced with CCl 4 . (A) Liver SOD activity, GSH levels, MDA levels and H 2 O 2 levels were measured. (B) Western blot analysis was used to determine SOD1, NQO-1, HO-1 and Nrf2 protein expression levels in the liver tissue samples. (C) p-p38, p-ERK1/2 and p-JNK protein levels in liver samples were calculated using western blot assays. Values are expressed as the mean ± standard error of the mean (n=8). ** P
Figure Legend Snippet: Breviscapine reduces oxidative stress in the livers of mice induced with CCl 4 . (A) Liver SOD activity, GSH levels, MDA levels and H 2 O 2 levels were measured. (B) Western blot analysis was used to determine SOD1, NQO-1, HO-1 and Nrf2 protein expression levels in the liver tissue samples. (C) p-p38, p-ERK1/2 and p-JNK protein levels in liver samples were calculated using western blot assays. Values are expressed as the mean ± standard error of the mean (n=8). ** P

Techniques Used: Mouse Assay, Activity Assay, Multiple Displacement Amplification, Western Blot, Expressing

Breviscapine reduces LPS-induced apoptosis in L02 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis was used to determine the levels of Bcl-2, Bax and Apaf-1 in LPS-treated L02 cells in the presence or absence of breviscapine. (B) Western blot analysis was performed to determine Bcl-2, Bax and Apaf-1 expression at the protein level. Representative western blot images and quantified protein levels are provided. (C) Apaf-1 positive cells were examined by immunofluorescence analysis among L02 cells exposed to LPS with or without breviscapine treatment (20 or 40 µ M). Scale bar, 50 µ m. (D) Active caspase-3 and PARP protein levels were assessed by western blot analysis. Representative western blot images and quantified protein levels are provided. Values are expressed as the mean ± standard error of the mean (n=10). ** P
Figure Legend Snippet: Breviscapine reduces LPS-induced apoptosis in L02 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis was used to determine the levels of Bcl-2, Bax and Apaf-1 in LPS-treated L02 cells in the presence or absence of breviscapine. (B) Western blot analysis was performed to determine Bcl-2, Bax and Apaf-1 expression at the protein level. Representative western blot images and quantified protein levels are provided. (C) Apaf-1 positive cells were examined by immunofluorescence analysis among L02 cells exposed to LPS with or without breviscapine treatment (20 or 40 µ M). Scale bar, 50 µ m. (D) Active caspase-3 and PARP protein levels were assessed by western blot analysis. Representative western blot images and quantified protein levels are provided. Values are expressed as the mean ± standard error of the mean (n=10). ** P

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence

Breviscapine improves CCl 4 -induced histological changes and collagen deposition in mouse livers. (A) Chemical structure of breviscapine. (B) Schematic of the study design. (C) Histological analysis of liver sections from CCl 4 -induced mice by hematoxylin and eosin staining, and the histological score. (D) Masson analysis for the deposition of collagen was performed. (E) Collagen accumulation was measured by Picric acid-Sirius red staining. Representative images and quantification are displayed. Scale bar, 100 µ m. Mean values are expressed as the mean ± standard error of the mean (n=10). **P
Figure Legend Snippet: Breviscapine improves CCl 4 -induced histological changes and collagen deposition in mouse livers. (A) Chemical structure of breviscapine. (B) Schematic of the study design. (C) Histological analysis of liver sections from CCl 4 -induced mice by hematoxylin and eosin staining, and the histological score. (D) Masson analysis for the deposition of collagen was performed. (E) Collagen accumulation was measured by Picric acid-Sirius red staining. Representative images and quantification are displayed. Scale bar, 100 µ m. Mean values are expressed as the mean ± standard error of the mean (n=10). **P

Techniques Used: Mouse Assay, Staining

Breviscapine ameliorates CCl 4 -induced liver injury by via suppression of TLR4/NF-κB signaling. (A) Immunofluorescence microscopy was performed to explore the TLR4 intensity in the liver sections of mice after CCl 4 induction. Scale bar, 100 µ m. (B) TLR4 and MyD88 protein levels were calculated by western blot analysis, displayed with the representative images and quantified levels. (C) Phosphorylated IKKα, IκBα and NF-κB levels were determined by immunoblot analysis. The quantified levels were also displayed. Values are expressed as the mean ± standard error of the mean (n=10). *** P
Figure Legend Snippet: Breviscapine ameliorates CCl 4 -induced liver injury by via suppression of TLR4/NF-κB signaling. (A) Immunofluorescence microscopy was performed to explore the TLR4 intensity in the liver sections of mice after CCl 4 induction. Scale bar, 100 µ m. (B) TLR4 and MyD88 protein levels were calculated by western blot analysis, displayed with the representative images and quantified levels. (C) Phosphorylated IKKα, IκBα and NF-κB levels were determined by immunoblot analysis. The quantified levels were also displayed. Values are expressed as the mean ± standard error of the mean (n=10). *** P

Techniques Used: Immunofluorescence, Microscopy, Mouse Assay, Western Blot

29) Product Images from "The Na+/H+ Exchanger Regulatory Factor Stabilizes Epidermal Growth Factor Receptors at the Cell Surface"

Article Title: The Na+/H+ Exchanger Regulatory Factor Stabilizes Epidermal Growth Factor Receptors at the Cell Surface

Journal:

doi: 10.1091/mbc.E04-03-0239

Interaction of EGFR with NHERF in the absence or presence of the tyrosine kinase inhibitor tyrphostin 1478. (A) Coimmunoprecipitation of endogenous EGFR and NHERF. HeLa and A431 cells were lysed and EGFRs were immunoprecipitated using the mouse monoclonal
Figure Legend Snippet: Interaction of EGFR with NHERF in the absence or presence of the tyrosine kinase inhibitor tyrphostin 1478. (A) Coimmunoprecipitation of endogenous EGFR and NHERF. HeLa and A431 cells were lysed and EGFRs were immunoprecipitated using the mouse monoclonal

Techniques Used: Immunoprecipitation

30) Product Images from "Lung Epithelial TRPA1 Transduces the Extracellular ROS into Transcriptional Regulation of Lung Inflammation Induced by Cigarette Smoke: The Role of Influxed Ca2+"

Article Title: Lung Epithelial TRPA1 Transduces the Extracellular ROS into Transcriptional Regulation of Lung Inflammation Induced by Cigarette Smoke: The Role of Influxed Ca2+

Journal: Mediators of Inflammation

doi: 10.1155/2015/148367

The CSE-induced extracellular ROS stimulate TRPA1 to Ca 2+ -dependently increase intracellular ROS via NADPH oxidase in HBECs. (a–c) Cells were exposed to medium alone or to 3% CSE for 2, 30, and 15 min, respectively, after pretreatment with apocynin (APO; an inhibitor of NADPH oxidase; 150 μ M), after pretreatment with N-acetyl-cysteine (NAC, a ROS scavenger; 1 mM), after pretreatment with HC-030031 (HC, a TRPA1 antagonist; 9 μ M), or after pretreatment with EGTA (an extracellular Ca 2+ chelator; 500 μ M). Levels of ROS were measured by HE fluorescent probe assay. NADPH oxidase activity was measured by NADP + /NADPH assay. Data in each group are mean ± SEM from five independent experiments. ∗ p
Figure Legend Snippet: The CSE-induced extracellular ROS stimulate TRPA1 to Ca 2+ -dependently increase intracellular ROS via NADPH oxidase in HBECs. (a–c) Cells were exposed to medium alone or to 3% CSE for 2, 30, and 15 min, respectively, after pretreatment with apocynin (APO; an inhibitor of NADPH oxidase; 150 μ M), after pretreatment with N-acetyl-cysteine (NAC, a ROS scavenger; 1 mM), after pretreatment with HC-030031 (HC, a TRPA1 antagonist; 9 μ M), or after pretreatment with EGTA (an extracellular Ca 2+ chelator; 500 μ M). Levels of ROS were measured by HE fluorescent probe assay. NADPH oxidase activity was measured by NADP + /NADPH assay. Data in each group are mean ± SEM from five independent experiments. ∗ p

Techniques Used: Activity Assay

31) Product Images from "Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes"

Article Title: Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes

Journal: Clinical Science (London, England : 1979)

doi: 10.1042/CS20100084

Effect of signalling pathway inhibitors on hypoxia-induced myocardin expression and ERK phosphorylation (A and B) ERK pathway mediates hypoxia-induced myocardin expression in neonatal cardiomyocytes. Neonatal cardiomyocytes were pre-treated with an ERK pathway inhibitor (PD98059), a JNK inhibitor (SP600125), a p38 MAPK inhibitor (SB203580), a PI3K/Akt inhibitor (wortmannin) or ERK siRNA, followed by hypoxia for 4 h. Neonatal cardiomyocytes were harvested and cell lysates were analysed by Western blotting using an anti-myocardin antibody. Result are normalized to actin levels. * P
Figure Legend Snippet: Effect of signalling pathway inhibitors on hypoxia-induced myocardin expression and ERK phosphorylation (A and B) ERK pathway mediates hypoxia-induced myocardin expression in neonatal cardiomyocytes. Neonatal cardiomyocytes were pre-treated with an ERK pathway inhibitor (PD98059), a JNK inhibitor (SP600125), a p38 MAPK inhibitor (SB203580), a PI3K/Akt inhibitor (wortmannin) or ERK siRNA, followed by hypoxia for 4 h. Neonatal cardiomyocytes were harvested and cell lysates were analysed by Western blotting using an anti-myocardin antibody. Result are normalized to actin levels. * P

Techniques Used: Expressing, Western Blot

32) Product Images from "Matrix metalloproteinase-1 up-regulation by hepatocyte growth factor in human dermal fibroblasts via ERK signaling pathway involves Ets1 and Fli1"

Article Title: Matrix metalloproteinase-1 up-regulation by hepatocyte growth factor in human dermal fibroblasts via ERK signaling pathway involves Ets1 and Fli1

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki648

Activation of ERK signaling pathway by HGF in human dermal fibroblasts. ( A ) Cell lysates (30 μg of protein/sample) were subjected to immunoblotting with anti-phospho-ERK antibody. That the amounts of ERK proteins were unchanged was confirmed by immunoblotting using anti-ERK2 antibodies. One experiment representative of three independent experiments is shown. The levels of phosphorylated ERK quantitated by scanning densitometry and corrected for the levels of total ERK in the same samples are shown relative to the level of untreated cells (1.0). Data are expressed as the mean ± SD of three independent experiments. ( B ) Human dermal fibroblasts were transiently transfected with the full-length MMP-1 promoter. After incubation overnight, the cells were pretreated with either vehicle alone (control), 10 or 30 μM LY294002, or 10 or 20 μM PD98059 for 1 h before the addition of HGF. Cells were incubated in the absence (open bars) or presence (closed bar) of 10 ng/ml HGF for additional 24 h. The graph depicts the MMP-1 promoter activities, and the basal promoter activity was arbitrarily set at 1. The number shows the promoter activities stimulated by HGF relative to the promoter without HGF. Data are expressed as the mean ± SD of three independent experiments. * P
Figure Legend Snippet: Activation of ERK signaling pathway by HGF in human dermal fibroblasts. ( A ) Cell lysates (30 μg of protein/sample) were subjected to immunoblotting with anti-phospho-ERK antibody. That the amounts of ERK proteins were unchanged was confirmed by immunoblotting using anti-ERK2 antibodies. One experiment representative of three independent experiments is shown. The levels of phosphorylated ERK quantitated by scanning densitometry and corrected for the levels of total ERK in the same samples are shown relative to the level of untreated cells (1.0). Data are expressed as the mean ± SD of three independent experiments. ( B ) Human dermal fibroblasts were transiently transfected with the full-length MMP-1 promoter. After incubation overnight, the cells were pretreated with either vehicle alone (control), 10 or 30 μM LY294002, or 10 or 20 μM PD98059 for 1 h before the addition of HGF. Cells were incubated in the absence (open bars) or presence (closed bar) of 10 ng/ml HGF for additional 24 h. The graph depicts the MMP-1 promoter activities, and the basal promoter activity was arbitrarily set at 1. The number shows the promoter activities stimulated by HGF relative to the promoter without HGF. Data are expressed as the mean ± SD of three independent experiments. * P

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

The binding activity of Ets1 or Fli1 is altered by HGF ( A ) To determine the amounts of Ets1 or Fli1 in cell lysates, human dermal fibroblasts were serum-starved for 24 h and treated with 10 ng/ml HGF for the indicated times. Immunoblotting were performed using anti-Ets1 or Fli1 antibodies. The same membrane was then stripped and reprobed with anti-β-actin antibody to show as a loading control. The levels of Ets1 (open bars) and Fli1 (closed bars) quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to those in untreated cells without HGF stimulation (1.0). Data are expressed as the mean ± SD of four experiments. ( B ) Human dermal fibroblasts were serum-starved for 24 h and pretreated with 10 or 20 μM PD98059 for 1 h before the addition of 10 ng/ml of HGF for 24 h. Cell lysates were subjected to immunoblotting with anti-Ets1 or Fli1 antibodies. ( C ) Nuclear extracts were prepared from dermal fibroblast and incubated with biotin-labeled oligonucleotide as described under ‘Materials and Methods’. Proteins bound to each nucleotide were isolated with streptavidin–agarose beads, and c-jun, Ets1 or Fli1 was detected by immunoblotting. The levels of Ets1 (open bars) and Fli1 (closed bars) quantitated by scanning densitometry are shown relative to the level of untreated cells (1.0). ( D ) Normal and SSc fibroblasts were incubated in serum-free medium for 24 h in the presence or absence of 10 ng/ml of HGF prior to collection of the cell lysates. Cell lysates (normalized for protein concentrations as measured with the Bio-Rad reagent) were subjected to immunoblotting with anti-Ets1 or Fli1 antibody. The same membrane was then stripped and reprobed with anti-β-actin antibody as a loading control. The representative results for two normal and two SSc fibroblasts are shown. ( E ) Ets1 (open bars) or Fli1 (close bars) levels quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to those in normal fibroblasts without HGF stimulation (1.0). Data are expressed as the mean ± SD of independent experiments. The number shows fold-stimulation with HGF relative to those without HGF in each cell type.
Figure Legend Snippet: The binding activity of Ets1 or Fli1 is altered by HGF ( A ) To determine the amounts of Ets1 or Fli1 in cell lysates, human dermal fibroblasts were serum-starved for 24 h and treated with 10 ng/ml HGF for the indicated times. Immunoblotting were performed using anti-Ets1 or Fli1 antibodies. The same membrane was then stripped and reprobed with anti-β-actin antibody to show as a loading control. The levels of Ets1 (open bars) and Fli1 (closed bars) quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to those in untreated cells without HGF stimulation (1.0). Data are expressed as the mean ± SD of four experiments. ( B ) Human dermal fibroblasts were serum-starved for 24 h and pretreated with 10 or 20 μM PD98059 for 1 h before the addition of 10 ng/ml of HGF for 24 h. Cell lysates were subjected to immunoblotting with anti-Ets1 or Fli1 antibodies. ( C ) Nuclear extracts were prepared from dermal fibroblast and incubated with biotin-labeled oligonucleotide as described under ‘Materials and Methods’. Proteins bound to each nucleotide were isolated with streptavidin–agarose beads, and c-jun, Ets1 or Fli1 was detected by immunoblotting. The levels of Ets1 (open bars) and Fli1 (closed bars) quantitated by scanning densitometry are shown relative to the level of untreated cells (1.0). ( D ) Normal and SSc fibroblasts were incubated in serum-free medium for 24 h in the presence or absence of 10 ng/ml of HGF prior to collection of the cell lysates. Cell lysates (normalized for protein concentrations as measured with the Bio-Rad reagent) were subjected to immunoblotting with anti-Ets1 or Fli1 antibody. The same membrane was then stripped and reprobed with anti-β-actin antibody as a loading control. The representative results for two normal and two SSc fibroblasts are shown. ( E ) Ets1 (open bars) or Fli1 (close bars) levels quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to those in normal fibroblasts without HGF stimulation (1.0). Data are expressed as the mean ± SD of independent experiments. The number shows fold-stimulation with HGF relative to those without HGF in each cell type.

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

Effects of LY294002 and PD98059 on the HGF-induced MMP-1 expression ( A ) Human dermal fibroblasts were serum-starved for 24 h and pretreated with 10 or 30 μM LY294002, or 10 or 20 μM PD98059, for 1 h before the addition of 10 ng/ml of HGF for 72 h. Conditioned medium were subjected to immunoblotting with anti-MMP-1 antibodies. ( B ) The northern blot analysis of MMP-1 mRNA expression was performed. LY294002 (30 μM) or PD98059 (20 μM) was added 1 h before the addition of 10 ng/ml of HGF. After 24 h, cells were collected. Levels of GAPDH mRNA are shown as a loading control. MMP-1 mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to the levels in untreated cells (1.0). One experiment representative of three independent experiments is shown. * P
Figure Legend Snippet: Effects of LY294002 and PD98059 on the HGF-induced MMP-1 expression ( A ) Human dermal fibroblasts were serum-starved for 24 h and pretreated with 10 or 30 μM LY294002, or 10 or 20 μM PD98059, for 1 h before the addition of 10 ng/ml of HGF for 72 h. Conditioned medium were subjected to immunoblotting with anti-MMP-1 antibodies. ( B ) The northern blot analysis of MMP-1 mRNA expression was performed. LY294002 (30 μM) or PD98059 (20 μM) was added 1 h before the addition of 10 ng/ml of HGF. After 24 h, cells were collected. Levels of GAPDH mRNA are shown as a loading control. MMP-1 mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to the levels in untreated cells (1.0). One experiment representative of three independent experiments is shown. * P

Techniques Used: Expressing, Northern Blot

33) Product Images from "Cross-regulation between protein L-isoaspartyl O-methyltransferase and ERK in epithelial mesenchymal transition of MDA-MB-231 cells"

Article Title: Cross-regulation between protein L-isoaspartyl O-methyltransferase and ERK in epithelial mesenchymal transition of MDA-MB-231 cells

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2011.94

The ERK inhibitor PD98059 alters the expression of PIMT and EMT proteins. (A, B, and C) Detached (d) MDA-MB-231 cells (10 6 cells/well ×6 wells) were maintained in suspension on poly-HEMA in serum-free media for indicated times in media containing PD98059 (PD, 25 μmol/L) or LiCl (Li, 2 μmol/L). After preparing whole lysates, the phosphorylation levels of ERK, MEK, Akt1, and GSK3 were analyzed by immunoblotting. (B) The phosphorylation levels of GSK3, ERK1/2, and p90RSK were determined by immunoblotting analysis of detached MDA-MB-231 cells (10 6 cells/well ×6 wells) cultured on poly-HEMA in the presence or absence of PD98059 (25 μmol/L) or LiCl (2 μmol/L) for 24 h. (C) The mRNA levels of fibronectin, TGF-β1, Slug, MMP-2, and MMP-9 were analyzed by RT-PCR using detached MDA-MB-231 cells (10 6 cells/well × 6 wells) cultured on poly-HEMA in the presence or absence of PD98059 (25 μmol/L) or LiCl (2 μmol/L) for 24 h. (D) The activity of MMP-2 and MMP-9 was examined by gelatin zymography using detached MDA-MB-231 cells (10 6 cells/well × 6 wells) cultured on poly-HEMA in the presence or absence of PD98059 (25 μmol/L) or LiCl (2 μmol/L) for 24 h.
Figure Legend Snippet: The ERK inhibitor PD98059 alters the expression of PIMT and EMT proteins. (A, B, and C) Detached (d) MDA-MB-231 cells (10 6 cells/well ×6 wells) were maintained in suspension on poly-HEMA in serum-free media for indicated times in media containing PD98059 (PD, 25 μmol/L) or LiCl (Li, 2 μmol/L). After preparing whole lysates, the phosphorylation levels of ERK, MEK, Akt1, and GSK3 were analyzed by immunoblotting. (B) The phosphorylation levels of GSK3, ERK1/2, and p90RSK were determined by immunoblotting analysis of detached MDA-MB-231 cells (10 6 cells/well ×6 wells) cultured on poly-HEMA in the presence or absence of PD98059 (25 μmol/L) or LiCl (2 μmol/L) for 24 h. (C) The mRNA levels of fibronectin, TGF-β1, Slug, MMP-2, and MMP-9 were analyzed by RT-PCR using detached MDA-MB-231 cells (10 6 cells/well × 6 wells) cultured on poly-HEMA in the presence or absence of PD98059 (25 μmol/L) or LiCl (2 μmol/L) for 24 h. (D) The activity of MMP-2 and MMP-9 was examined by gelatin zymography using detached MDA-MB-231 cells (10 6 cells/well × 6 wells) cultured on poly-HEMA in the presence or absence of PD98059 (25 μmol/L) or LiCl (2 μmol/L) for 24 h.

Techniques Used: Expressing, Multiple Displacement Amplification, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Zymography

34) Product Images from "P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells"

Article Title: P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

Journal: Immunology

doi: 10.1111/imm.12886

The deletion of Pak2 in peripheral CD4 T cells inhibits the phosphorylation of p70S6K, S6 and cofilin. Pak2 F/F ; Cd4‐ ER T2 ‐Cre; Rosa26 ‐YFP or wild‐type (WT) control ( Pak2 F/F ) mice were treated with tamoxifen for 5 days and rested for another 5 days. CD4 + CD25 − T cells cells from WT or inducible Pak2 knockout (KO) mice were isolated and then stimulated in vitro with CD3 stimulation (10 μg/ml, pre‐coated) in the presence or absence of CD28 co‐stimulation (10 μg/ml, pre‐coated) for 5 min or 30 min. The cell lysates were prepared, and the phosphorylation levels of p70S6K, S6K and cofilin were measured by Western blot analysis. GAPDH was used as a loading control. Molecular weight markers are indicated. All results are representative of two independent experiments.
Figure Legend Snippet: The deletion of Pak2 in peripheral CD4 T cells inhibits the phosphorylation of p70S6K, S6 and cofilin. Pak2 F/F ; Cd4‐ ER T2 ‐Cre; Rosa26 ‐YFP or wild‐type (WT) control ( Pak2 F/F ) mice were treated with tamoxifen for 5 days and rested for another 5 days. CD4 + CD25 − T cells cells from WT or inducible Pak2 knockout (KO) mice were isolated and then stimulated in vitro with CD3 stimulation (10 μg/ml, pre‐coated) in the presence or absence of CD28 co‐stimulation (10 μg/ml, pre‐coated) for 5 min or 30 min. The cell lysates were prepared, and the phosphorylation levels of p70S6K, S6K and cofilin were measured by Western blot analysis. GAPDH was used as a loading control. Molecular weight markers are indicated. All results are representative of two independent experiments.

Techniques Used: Mouse Assay, Knock-Out, Isolation, In Vitro, Western Blot, Molecular Weight

35) Product Images from "Hypoxia-Regulated Delta-like 1 Homologue Enhances Cancer Cell Stemness and Tumorigenicity"

Article Title: Hypoxia-Regulated Delta-like 1 Homologue Enhances Cancer Cell Stemness and Tumorigenicity

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-09-1605

DLK1 maintains an undifferentiated NB phenotype. A, BE(2)C cells were differentiated with 1 μmol/L RA or 10 μmol/L BrdUrd for 5 d. DLK1, Sox2, c-kit, and CD-133 were detected by Western blot. B, BE(2)C cells were infected with lentivirus
Figure Legend Snippet: DLK1 maintains an undifferentiated NB phenotype. A, BE(2)C cells were differentiated with 1 μmol/L RA or 10 μmol/L BrdUrd for 5 d. DLK1, Sox2, c-kit, and CD-133 were detected by Western blot. B, BE(2)C cells were infected with lentivirus

Techniques Used: Western Blot, Infection

36) Product Images from "Focal Adhesion Kinase Is Required, But Not Sufficient, for the Induction of Long-Term Potentiation in Dentate Gyrus Neurons In Vivo"

Article Title: Focal Adhesion Kinase Is Required, But Not Sufficient, for the Induction of Long-Term Potentiation in Dentate Gyrus Neurons In Vivo

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.23-10-04072.2003

Tetanic stimulation enhances the association of FAK[pY 397 ] with NR2A and NR2B as well as the increased tyrosine phosphorylation of NR2A and NR2B. A , Precleared synaptosomal fraction (100 μg) from dentate gyrus was immunoprecipitated with anti-FAK[pY 397 ] antibody, followed by immunoblotting with anti-NR1, NR2A, NR2B, PI3-kinase, and FAK antibodies. Increased association with NR2A and NR2B was observed with tetanic stimulation. This association was decreased in the HA-FAK(Y397F)-transfected group. The association of FAK[pY 397 ] with NR1 and PI3-kinase remained similar. B , Immunoprecipitation was performed by using anti-NR2A and anti-NR2B antibodies, followed by immunoblotting with anti-FAK[pY 397 ], FAK, phosphotyrosine, NR2A, and NR2B antibodies. Similarly, the association of FAK[pY 397 ] with NR2A and NR2B as well as the tyrosine phosphorylation of NR2A and NR2B was increased with tetanic stimulation. This increased association was prevented by HA-FAK(Y397F) transfection. The association of nonphosphorylated FAK with NR2A and NR2B was unchanged; n = 3–5 for each group.
Figure Legend Snippet: Tetanic stimulation enhances the association of FAK[pY 397 ] with NR2A and NR2B as well as the increased tyrosine phosphorylation of NR2A and NR2B. A , Precleared synaptosomal fraction (100 μg) from dentate gyrus was immunoprecipitated with anti-FAK[pY 397 ] antibody, followed by immunoblotting with anti-NR1, NR2A, NR2B, PI3-kinase, and FAK antibodies. Increased association with NR2A and NR2B was observed with tetanic stimulation. This association was decreased in the HA-FAK(Y397F)-transfected group. The association of FAK[pY 397 ] with NR1 and PI3-kinase remained similar. B , Immunoprecipitation was performed by using anti-NR2A and anti-NR2B antibodies, followed by immunoblotting with anti-FAK[pY 397 ], FAK, phosphotyrosine, NR2A, and NR2B antibodies. Similarly, the association of FAK[pY 397 ] with NR2A and NR2B as well as the tyrosine phosphorylation of NR2A and NR2B was increased with tetanic stimulation. This increased association was prevented by HA-FAK(Y397F) transfection. The association of nonphosphorylated FAK with NR2A and NR2B was unchanged; n = 3–5 for each group.

Techniques Used: Immunoprecipitation, Transfection

37) Product Images from "ERF Nuclear Shuttling, a Continuous Monitor of Erk Activity That Links It to Cell Cycle Progression"

Article Title: ERF Nuclear Shuttling, a Continuous Monitor of Erk Activity That Links It to Cell Cycle Progression

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.3.1206-1218.2004

ERF is phosphorylated in the nucleus. (A) Ref cells were subjected to serum arrest for 2 h and consequent serum stimulation for the indicated times. The localization of the phosphorylated ERF was determined by indirect immunofluorescence using the P3-4 phospho-specific anti-ERF antibody (green), and that of total ERF was determined with the S17S anti-ERF antibody (red). Nuclei ware stained blue by TO-PRO-3 (left). At the same time points, activated Erks were detected with the phospho-specific anti-Erk monoclonal antibody MAPK-YT (green). Nuclei ware stained blue by TO-PRO-3 (right). Red and blue color colocalization shows as magenta, green and blue colocalization shows as cyan, green and red colocalization shows as yellow, and colocalization of all three shows as white. (B) Under the same conditions as in panel A, total cell extract was analyzed by immunoblotting for total and phosphorylated ERF and Erks, as indicated. The S17S, P3-4, P7, MAPK-YT, and anti-Erk specific polyclonal antibodies were used to determine total ERF, ERF phosphorylated at S246 and S251, ERF phosphorylated at T526, phosphorylated Erks, and total Erks, respectively. FBS, fetal bovine serum. (C) Ref cells were deprived of serum for 1 h and then induced with serum. At the indicated times cells were harvested and separated into nuclear and cytoplasmic fractions. The amount of the phosphorylated ERF protein in each fraction was determined by immunoblotting. Both the P3-4 (top) and the P7 (bottom) phospho-specific anti-ERF antibodies were used. (D) Ref cells were serum arrested for 2 h and then treated with the indicated amounts of epidermal growth factor for 5 min. Total cell extracts were analyzed for total and phosphorylated ERF and Erks, as for panel B.
Figure Legend Snippet: ERF is phosphorylated in the nucleus. (A) Ref cells were subjected to serum arrest for 2 h and consequent serum stimulation for the indicated times. The localization of the phosphorylated ERF was determined by indirect immunofluorescence using the P3-4 phospho-specific anti-ERF antibody (green), and that of total ERF was determined with the S17S anti-ERF antibody (red). Nuclei ware stained blue by TO-PRO-3 (left). At the same time points, activated Erks were detected with the phospho-specific anti-Erk monoclonal antibody MAPK-YT (green). Nuclei ware stained blue by TO-PRO-3 (right). Red and blue color colocalization shows as magenta, green and blue colocalization shows as cyan, green and red colocalization shows as yellow, and colocalization of all three shows as white. (B) Under the same conditions as in panel A, total cell extract was analyzed by immunoblotting for total and phosphorylated ERF and Erks, as indicated. The S17S, P3-4, P7, MAPK-YT, and anti-Erk specific polyclonal antibodies were used to determine total ERF, ERF phosphorylated at S246 and S251, ERF phosphorylated at T526, phosphorylated Erks, and total Erks, respectively. FBS, fetal bovine serum. (C) Ref cells were deprived of serum for 1 h and then induced with serum. At the indicated times cells were harvested and separated into nuclear and cytoplasmic fractions. The amount of the phosphorylated ERF protein in each fraction was determined by immunoblotting. Both the P3-4 (top) and the P7 (bottom) phospho-specific anti-ERF antibodies were used. (D) Ref cells were serum arrested for 2 h and then treated with the indicated amounts of epidermal growth factor for 5 min. Total cell extracts were analyzed for total and phosphorylated ERF and Erks, as for panel B.

Techniques Used: Immunofluorescence, Staining

38) Product Images from "Epoxyeicosatrienoic acid-stimulated proliferation in cancer cells involves EGF-R phosphorylation mediated by activation of metalloproteinases and release of HB-EGF"

Article Title: Epoxyeicosatrienoic acid-stimulated proliferation in cancer cells involves EGF-R phosphorylation mediated by activation of metalloproteinases and release of HB-EGF

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2009.184

Effects of AG1478, CRM197, and 1,10-phenanthroline on the 14,15-EET-induced tyrosine phosphorylation of EGF-R and ERK1/2 in the A549, HepG2, and MDA-MB-231 cell lines. The upper panel is representative of three separate experiments with similar results.
Figure Legend Snippet: Effects of AG1478, CRM197, and 1,10-phenanthroline on the 14,15-EET-induced tyrosine phosphorylation of EGF-R and ERK1/2 in the A549, HepG2, and MDA-MB-231 cell lines. The upper panel is representative of three separate experiments with similar results.

Techniques Used: Multiple Displacement Amplification

Effects of 1,10-phenanthroline and CRM197 on the 14,15-EET-induced tyrosine phosphorylation of EGF-R and ERK1/2 in Tca-8113 cells. Serum-deprived Tca-8113 cells were incubated with or without 100 μmol/L of 1,10-phenanthroline or 10 μg/mL
Figure Legend Snippet: Effects of 1,10-phenanthroline and CRM197 on the 14,15-EET-induced tyrosine phosphorylation of EGF-R and ERK1/2 in Tca-8113 cells. Serum-deprived Tca-8113 cells were incubated with or without 100 μmol/L of 1,10-phenanthroline or 10 μg/mL

Techniques Used: Incubation

Effects of tyrphostin AG1478, 1,10-phenanthroline, and the nontoxic mutant of diphtheria toxin CRM197 on the activation of EGF-R and ERK1/2 in rAAV-CYP102 F87V-transfected Tca-8113 cells. Tca-8113 cells were transfected with rAAV-CYP102 F87V or rAAV-GFP
Figure Legend Snippet: Effects of tyrphostin AG1478, 1,10-phenanthroline, and the nontoxic mutant of diphtheria toxin CRM197 on the activation of EGF-R and ERK1/2 in rAAV-CYP102 F87V-transfected Tca-8113 cells. Tca-8113 cells were transfected with rAAV-CYP102 F87V or rAAV-GFP

Techniques Used: Mutagenesis, Activation Assay, Transfection

39) Product Images from "c-Jun N-Terminal Protein Kinase 1 (JNK1), but Not JNK2, Is Essential for Tumor Necrosis Factor Alpha-Induced c-Jun Kinase Activation and Apoptosis"

Article Title: c-Jun N-Terminal Protein Kinase 1 (JNK1), but Not JNK2, Is Essential for Tumor Necrosis Factor Alpha-Induced c-Jun Kinase Activation and Apoptosis

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.24.10844-10856.2004

JNK2 interferes with JNK1 activation. Cells were stimulated (+) with TNF-α (5 ng/ml, 15 min) (A), UV (60 J/m 2 , 30 min) and anisomycin (Aniso.) (50 ng/ml, 30 min) (B), and IL-1β (2 ng/ml, 15 min) and TPA (100 ng/ml, 30 min) (C). Endogenous JNK1 was isolated by using the anti-JNK antibody (α-JNK) (antibody 333; PharMingen), and its activity was measured by immune complex kinase assays (KA). The levels of JNK isoforms expressed were measured by immunoblotting (IB) with an anti-JNK antibody (antibody 666; PharMingen). The levels of protein expressed and activation of p38 and ERK were analyzed by immunoblotting with the appropriate antibodies. P-p38, phosphorylated p38. (D) Phosphorylation of endogenous JNK isoforms was analyzed by immunoblotting with anti-phospho-JNK antibody (α-P-JNK). The levels of JNK expressed were analyzed by immunoblotting with the anti-JNK antibody (antibody 666; PharMingen). (E) Jnk2 − / − cells were transfected (+) with expression vectors encoding M2-JNK1, HA-JNK2, the kinase-deficient HA-JNK2(KM) mutant, or empty vector (2 μg each) and stimulated with TNF-α (5 ng/ml, 15 min) (+) or left alone. The activities of M2-JNK1 and HA-JNK2, as well as expression of M2-JNK1 and HA-JNK2, were analyzed by immune complex kinase assays (KA) and immunoblotting (IB), respectively. (F) Cells were transfected with an expression vector encoding HA-JNK2 (2 μg) (+) and stimulated with TNF-α (5 ng/ml, 15 min) (+) or not stimulated with TNF-α (−). The activity and expression of HA-JNK2 were measured above as described for panel E.
Figure Legend Snippet: JNK2 interferes with JNK1 activation. Cells were stimulated (+) with TNF-α (5 ng/ml, 15 min) (A), UV (60 J/m 2 , 30 min) and anisomycin (Aniso.) (50 ng/ml, 30 min) (B), and IL-1β (2 ng/ml, 15 min) and TPA (100 ng/ml, 30 min) (C). Endogenous JNK1 was isolated by using the anti-JNK antibody (α-JNK) (antibody 333; PharMingen), and its activity was measured by immune complex kinase assays (KA). The levels of JNK isoforms expressed were measured by immunoblotting (IB) with an anti-JNK antibody (antibody 666; PharMingen). The levels of protein expressed and activation of p38 and ERK were analyzed by immunoblotting with the appropriate antibodies. P-p38, phosphorylated p38. (D) Phosphorylation of endogenous JNK isoforms was analyzed by immunoblotting with anti-phospho-JNK antibody (α-P-JNK). The levels of JNK expressed were analyzed by immunoblotting with the anti-JNK antibody (antibody 666; PharMingen). (E) Jnk2 − / − cells were transfected (+) with expression vectors encoding M2-JNK1, HA-JNK2, the kinase-deficient HA-JNK2(KM) mutant, or empty vector (2 μg each) and stimulated with TNF-α (5 ng/ml, 15 min) (+) or left alone. The activities of M2-JNK1 and HA-JNK2, as well as expression of M2-JNK1 and HA-JNK2, were analyzed by immune complex kinase assays (KA) and immunoblotting (IB), respectively. (F) Cells were transfected with an expression vector encoding HA-JNK2 (2 μg) (+) and stimulated with TNF-α (5 ng/ml, 15 min) (+) or not stimulated with TNF-α (−). The activity and expression of HA-JNK2 were measured above as described for panel E.

Techniques Used: Activation Assay, Isolation, Activity Assay, Immune Complex Kinase Assay, Transfection, Expressing, Mutagenesis, Plasmid Preparation

40) Product Images from "Fexaramine Inhibits Receptor Activator of Nuclear Factor-κB Ligand-induced Osteoclast Formation via Nuclear Factor of Activated T Cells Signaling Pathways"

Article Title: Fexaramine Inhibits Receptor Activator of Nuclear Factor-κB Ligand-induced Osteoclast Formation via Nuclear Factor of Activated T Cells Signaling Pathways

Journal: Journal of Bone Metabolism

doi: 10.11005/jbm.2017.24.4.207

Fexaramine (Fexa) inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced expression of c-Fos and nuclear factor of activated T cells (NFATc1). (A, B) Bone marrow-derived macrophages (BMMs) were preincubated in the absence or presence of fexa (5 µM) for 30 min, and then treated with or without 200 ng/mL of RANKL for 24 hr. Cell lysates were then subjected to Western blot analysis using (A) NFATc1 or (B) c-Fos antibodies. (C) BMMs were infected through the retrovirus packaging system. Infected BMMs were cultured with RANKL (100 ng/mL) and macrophage-colony stimulating factor (30 ng/mL) in the absence or presence of fexa (5 µM) for 4 days. The recovery rate was defined as the percentage of osteoclast formation in the presence of fexa. The osteoclast formation in the presence of vehicle was given as 100%. Data are expressed as mean±standard deviation from at least three independent experiments. * P
Figure Legend Snippet: Fexaramine (Fexa) inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced expression of c-Fos and nuclear factor of activated T cells (NFATc1). (A, B) Bone marrow-derived macrophages (BMMs) were preincubated in the absence or presence of fexa (5 µM) for 30 min, and then treated with or without 200 ng/mL of RANKL for 24 hr. Cell lysates were then subjected to Western blot analysis using (A) NFATc1 or (B) c-Fos antibodies. (C) BMMs were infected through the retrovirus packaging system. Infected BMMs were cultured with RANKL (100 ng/mL) and macrophage-colony stimulating factor (30 ng/mL) in the absence or presence of fexa (5 µM) for 4 days. The recovery rate was defined as the percentage of osteoclast formation in the presence of fexa. The osteoclast formation in the presence of vehicle was given as 100%. Data are expressed as mean±standard deviation from at least three independent experiments. * P

Techniques Used: Expressing, Derivative Assay, Western Blot, Infection, Cell Culture, Standard Deviation

Fexaramine (Fexa) inhibits bone resorption. (A) Fexa (5 µM) was added during the indicated culture days in the presence of macrophage colony-stimulating factor (M-CSF) (30 ng/mL) and receptor activator of nuclear factor-κB ligand (RANKL) (100 ng/mL). (B) Bone marrow-derived macrophages (BMMs) from farnesoid X receptor (FXR) +/+ and FXR −/− mice were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of indicated concentrations of fexa for 4 days, and tartrate-resistant acid phosphatase-positive (TRAP + ) osteoclasts were counted. (C) BMMs were differentiated on dentine slices with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for four days and fexa (5 µM) was treated for an additional two days. The number of resorption pits were counted. Scale bar=200 µm. Data are expressed as mean±standard deviation from at least three independent experiments. * P
Figure Legend Snippet: Fexaramine (Fexa) inhibits bone resorption. (A) Fexa (5 µM) was added during the indicated culture days in the presence of macrophage colony-stimulating factor (M-CSF) (30 ng/mL) and receptor activator of nuclear factor-κB ligand (RANKL) (100 ng/mL). (B) Bone marrow-derived macrophages (BMMs) from farnesoid X receptor (FXR) +/+ and FXR −/− mice were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of indicated concentrations of fexa for 4 days, and tartrate-resistant acid phosphatase-positive (TRAP + ) osteoclasts were counted. (C) BMMs were differentiated on dentine slices with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for four days and fexa (5 µM) was treated for an additional two days. The number of resorption pits were counted. Scale bar=200 µm. Data are expressed as mean±standard deviation from at least three independent experiments. * P

Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Standard Deviation

Fexaramine (Fexa) inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. (A) Bone marrow-derived macrophages (BMMs) were cultured with RANKL (100 ng/mL) and macrophage colony-stimulating factor (M-CSF) (30 ng/mL) in the presence of the indicated concentration of fexa for 4 days and tartrate-resistant acid phosphatase-positive (TRAP + ) multinucleated osteoclasts (MNCs) were counted. (B) BMMs were cultured with M-CSF (30 ng/mL) in the presence or absence of fexa (5 µM) for 2 days and an microtitration assay was performed. (C) BMMs were cultured with M-CSF (30 ng/mL) in the presence or absence of fexa (5 µM) for 4 days and messenger ribonucleic acids expression level was determined by real time-polymerase chain reaction. Data are expressed as mean±standard deviation from at least three independent experiments. * P
Figure Legend Snippet: Fexaramine (Fexa) inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. (A) Bone marrow-derived macrophages (BMMs) were cultured with RANKL (100 ng/mL) and macrophage colony-stimulating factor (M-CSF) (30 ng/mL) in the presence of the indicated concentration of fexa for 4 days and tartrate-resistant acid phosphatase-positive (TRAP + ) multinucleated osteoclasts (MNCs) were counted. (B) BMMs were cultured with M-CSF (30 ng/mL) in the presence or absence of fexa (5 µM) for 2 days and an microtitration assay was performed. (C) BMMs were cultured with M-CSF (30 ng/mL) in the presence or absence of fexa (5 µM) for 4 days and messenger ribonucleic acids expression level was determined by real time-polymerase chain reaction. Data are expressed as mean±standard deviation from at least three independent experiments. * P

Techniques Used: Derivative Assay, Cell Culture, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Fexaramine (Fexa) inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced signaling pathways. (A-C) Bone marrow-derived macrophages (BMMs) were preincubated in the absence or presence of fexa (5 µM) for 30 min, and then treated with or without 200 ng/mL RANKL for (A, B) 15 min or (C) 24 hr. Cell lysates were then subjected to Western blotting analysis with the indicated antibodies. Data are expressed as the mean±standard deviation from at least three independent experiments. * P
Figure Legend Snippet: Fexaramine (Fexa) inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced signaling pathways. (A-C) Bone marrow-derived macrophages (BMMs) were preincubated in the absence or presence of fexa (5 µM) for 30 min, and then treated with or without 200 ng/mL RANKL for (A, B) 15 min or (C) 24 hr. Cell lysates were then subjected to Western blotting analysis with the indicated antibodies. Data are expressed as the mean±standard deviation from at least three independent experiments. * P

Techniques Used: Derivative Assay, Western Blot, Standard Deviation

Fexaramine (Fexa) suppresses lipopolysaccharide (LPS)-induced osteoclast formation in vivo. (A) Calvaria of mice that received vehicle, LPS, or LPS plus fexa (5 mg/kg) were subjected to tartrate-resistant acid phosphatase (TRAP) staining. (B) TRAP+stained area in calvaria were quantified using the image J program. Representative images are shown. Data are expressed as the mean±standard deviation from at least three independent experiments. * P
Figure Legend Snippet: Fexaramine (Fexa) suppresses lipopolysaccharide (LPS)-induced osteoclast formation in vivo. (A) Calvaria of mice that received vehicle, LPS, or LPS plus fexa (5 mg/kg) were subjected to tartrate-resistant acid phosphatase (TRAP) staining. (B) TRAP+stained area in calvaria were quantified using the image J program. Representative images are shown. Data are expressed as the mean±standard deviation from at least three independent experiments. * P

Techniques Used: In Vivo, Mouse Assay, Staining, Standard Deviation

Related Articles

Blocking Assay:

Article Title: Signalling through AMPA receptors on oligodendrocyte precursors promotes myelination by enhancing oligodendrocyte survival
Article Snippet: .. In some experiments the following drugs were applied by adding to the superfusing extracellular solution: kainic acid monohydrate (100 µM; Sigma) to activate AMPAR and kainate receptors, GYKI52466 (GYKI; 50 µM; Sigma) to block AMPARs, or RR (100 µM; Sigma) to evoke transmitter release. .. To record the OP’s voltage-gated currents and the I–V relationship of drug-evoked currents, 20 mV voltage steps of 200 ms duration were applied from a holding potential of –63 mV (voltage range from –103 mV to +17 mV).

Cell Surface Biotinylation Assay:

Article Title: Modulation of Synaptic Plasticity by Antimanic Agents: The Role of AMPA Glutamate Receptor Subunit 1 Synaptic Expression
Article Snippet: .. Surface GluR1 receptors were detected by a biotinylation assay, followed by Western blot analysis that used either a GluR1 antibody (1:200; Chemicon) or a pan-cadherin antibody (1:500; Sigma, St. Louis, MO). .. At the end of valproate or lithium treatment for the time indicated, ice-cold PBS (with calcium and magnesium, pH 7.4; Invitrogen) was added to the cultures to prevent receptor internalization.

Concentration Assay:

Article Title: CYR61/CCN1 and WISP3/CCN6 are chemoattractive ligands for human multipotent mesenchymal stroma cells
Article Snippet: .. Finally, the involvement of integrin receptors ανß 3 and ανß 5 was studied by preincubation of MSCs with specific antibodies (LM609 for ανß 3 and P1F6 for ανß 5, Chemicon International, United Kingdom) for 1 h at a concentration of 50 μg/ml each. .. Preincubation with 50 μg/ml normal mouse IgG (Sigma, Deisenhofen, Germany) was used as an unspecific control.

Incubation:

Article Title: The type I TGF-? receptor is covalently modified and regulated by sumoylation
Article Snippet: .. Immunopurified receptors were incubated at 30°C for 30 min in 10 mM HEPES-KOH, pH 7.5, 5 mM MgCl2 and 5 mM CaCl2 with or without 5 μM TβRI kinase inhibitor SB431542 (Sigma-Aldrich). .. The reaction mixture was subjected to SDS-PAGE, followed by autoradiography.

Western Blot:

Article Title: Modulation of Synaptic Plasticity by Antimanic Agents: The Role of AMPA Glutamate Receptor Subunit 1 Synaptic Expression
Article Snippet: .. Surface GluR1 receptors were detected by a biotinylation assay, followed by Western blot analysis that used either a GluR1 antibody (1:200; Chemicon) or a pan-cadherin antibody (1:500; Sigma, St. Louis, MO). .. At the end of valproate or lithium treatment for the time indicated, ice-cold PBS (with calcium and magnesium, pH 7.4; Invitrogen) was added to the cultures to prevent receptor internalization.

Staining:

Article Title: State-Dependent AMPA Receptor Trafficking in the Mammalian Retina
Article Snippet: .. For surface staining of GluR2-containing receptors, an antibody recognizing the extracellular N terminus of GluR2 subunit (7.5 μg/ml; Chemicon) was applied to live cells for 30 min at 37°C in complete medium. .. Cells were then fixed at indicated times with 4% paraformaldehyde in PBS for 10 min. After TBS wash, cells were blocked with 2% BSC in TBS and incubated with donkey anti-mouse Cy3 (5 μg/ml; Jackson ImmunoResearch) for 45 min. After several washes, slips were mounted in Vectashield.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Millipore phospho specific extracellular signal regulated kinase
    Phospho Specific Extracellular Signal Regulated Kinase, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho specific extracellular signal regulated kinase/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho specific extracellular signal regulated kinase - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    Image Search Results