Structured Review

Millipore u0126
EGFR/ERK signaling pathway is involved in the CsA-induced proliferation of human trophoblast cells. A : Primary human trophoblast cells and JEG-3 cells were treated with 1 μM CsA and neutralizing antibody against CXCR4 (20 µg/ml) or CXCL12 (40 µg/ml), or <t>U0126</t> (20 μM), or LY294002 (20 μM), or AG1478 (200 nM) for 48 h, and then subjected to BrdU cell proliferation assay. Data are presented as mean ± SEM of three independent experiments. *P
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Images

1) Product Images from "CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells"

Article Title: CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038375

EGFR/ERK signaling pathway is involved in the CsA-induced proliferation of human trophoblast cells. A : Primary human trophoblast cells and JEG-3 cells were treated with 1 μM CsA and neutralizing antibody against CXCR4 (20 µg/ml) or CXCL12 (40 µg/ml), or U0126 (20 μM), or LY294002 (20 μM), or AG1478 (200 nM) for 48 h, and then subjected to BrdU cell proliferation assay. Data are presented as mean ± SEM of three independent experiments. *P
Figure Legend Snippet: EGFR/ERK signaling pathway is involved in the CsA-induced proliferation of human trophoblast cells. A : Primary human trophoblast cells and JEG-3 cells were treated with 1 μM CsA and neutralizing antibody against CXCR4 (20 µg/ml) or CXCL12 (40 µg/ml), or U0126 (20 μM), or LY294002 (20 μM), or AG1478 (200 nM) for 48 h, and then subjected to BrdU cell proliferation assay. Data are presented as mean ± SEM of three independent experiments. *P

Techniques Used: BrdU Cell Proliferation Assay

2) Product Images from "Experience-Driven Axon Retraction in the Pharmacologically Inactivated Visual Cortex Does Not Require Synaptic Transmission"

Article Title: Experience-Driven Axon Retraction in the Pharmacologically Inactivated Visual Cortex Does Not Require Synaptic Transmission

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004193

Example of labeled axons in BoNT/E-treated visual cortex. (A) Low-magnification image of coronal section across visual cortex of BoNT/E-injected kitten. An arrowhead indicates the BoNT/E injection site. (B, C) High-magnification view of two regions in A far from (dotted line, B) and close to (solid line, C) the injection site. Egr-1 signal is detectable in the area far from the injection site (B), whereas the signal is very low in the area near the injection site (C). The staining at the lower-left in C represents labeled axons. (D) Enlarged view of geniculocortical axons in BoNT/E inactivated area (box in C). Scale bar, A: 1 mm, B, C: 100 µm, D: 10 µm.
Figure Legend Snippet: Example of labeled axons in BoNT/E-treated visual cortex. (A) Low-magnification image of coronal section across visual cortex of BoNT/E-injected kitten. An arrowhead indicates the BoNT/E injection site. (B, C) High-magnification view of two regions in A far from (dotted line, B) and close to (solid line, C) the injection site. Egr-1 signal is detectable in the area far from the injection site (B), whereas the signal is very low in the area near the injection site (C). The staining at the lower-left in C represents labeled axons. (D) Enlarged view of geniculocortical axons in BoNT/E inactivated area (box in C). Scale bar, A: 1 mm, B, C: 100 µm, D: 10 µm.

Techniques Used: Labeling, Injection, Staining

Effects of BoNT/E on cortical activity and Egr-1 expression level in visual cortex. (A) Examples of spike activity in rat visual cortex contralateral (upper trace, Control) and ipsilateral (lower trace, BoNT/E) to BoNT/E injection site recorded 1 week after injection. BoNT/E blocked neuronal activity only in the injected cortex. (B) Egr-1 expression level is significantly decreased in the BoNT/E injected cortex. The upper right panel shows examples of immunoblots for Egr-1 in the visual cortex of normal rats (Norm) and that 1 week after BoNT/E injection. Blot densities were normalized to those of GAPDH and expressed as the ratio to those of Egr-1 in normal animals. Error bars indicate SEM. *: P
Figure Legend Snippet: Effects of BoNT/E on cortical activity and Egr-1 expression level in visual cortex. (A) Examples of spike activity in rat visual cortex contralateral (upper trace, Control) and ipsilateral (lower trace, BoNT/E) to BoNT/E injection site recorded 1 week after injection. BoNT/E blocked neuronal activity only in the injected cortex. (B) Egr-1 expression level is significantly decreased in the BoNT/E injected cortex. The upper right panel shows examples of immunoblots for Egr-1 in the visual cortex of normal rats (Norm) and that 1 week after BoNT/E injection. Blot densities were normalized to those of GAPDH and expressed as the ratio to those of Egr-1 in normal animals. Error bars indicate SEM. *: P

Techniques Used: Activity Assay, Expressing, Injection, Western Blot

3) Product Images from "Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton"

Article Title: Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126214

Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P
Figure Legend Snippet: Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P

Techniques Used: Clone Assay, Generated, Stable Transfection, Isolation, SDS Page, Western Blot, Whisker Assay

4) Product Images from "Helicobacter Pylori Promotes the Expression of Kr?ppel-Like Factor 5, a Mediator of Carcinogenesis, In Vitro and In Vivo"

Article Title: Helicobacter Pylori Promotes the Expression of Kr?ppel-Like Factor 5, a Mediator of Carcinogenesis, In Vitro and In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054344

H. pylori induces expansion of a KLF5 + cell population in vivo . (A–G) KLF5 expression in murine gastric epithelial cells was assessed by flow cytometry analysis in uninfected and H. pylori -infected mice at acute time points (24, 48, 72 hours, and 1 week) and chronic time points (4 and 8 weeks) post-challenge. Percentage of KLF5 + cells at 4 weeks (A) and 8 weeks (C) and levels of KLF5 protein at 4 weeks (B) and 8 weeks (D), as determined by mean fluorescence units (MFU), were determined by flow cytometry. Data from 4 and 8 week time points were analyzed at separate times. H. pylori colonization density in mice infected for 24, 48, and 72 hours, and 1 week was assessed by quantitative culture (E). Percentage of KLF5 + cells (F) and levels of KLF5 protein (G) at 24, 48, or 72 hours, or 1 week were determined by flow cytometry. Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
Figure Legend Snippet: H. pylori induces expansion of a KLF5 + cell population in vivo . (A–G) KLF5 expression in murine gastric epithelial cells was assessed by flow cytometry analysis in uninfected and H. pylori -infected mice at acute time points (24, 48, 72 hours, and 1 week) and chronic time points (4 and 8 weeks) post-challenge. Percentage of KLF5 + cells at 4 weeks (A) and 8 weeks (C) and levels of KLF5 protein at 4 weeks (B) and 8 weeks (D), as determined by mean fluorescence units (MFU), were determined by flow cytometry. Data from 4 and 8 week time points were analyzed at separate times. H. pylori colonization density in mice infected for 24, 48, and 72 hours, and 1 week was assessed by quantitative culture (E). Percentage of KLF5 + cells (F) and levels of KLF5 protein (G) at 24, 48, or 72 hours, or 1 week were determined by flow cytometry. Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

Techniques Used: In Vivo, Expressing, Flow Cytometry, Cytometry, Infection, Mouse Assay, Fluorescence, MANN-WHITNEY

KLF5 and Ki67 co-localize to the isthmal region. KLF5 and Ki67 immunohistochemistry staining was assessed on murine gastric tissue sections from uninfected mice (A and C) or H. pylori PMSS1-infected mice (B and D) at 400× magnification. Insets demonstrate regions of KLF5 and Ki67 co-localization (arrows) within the isthmal regions of the gastric epithelium (E and F). Nuclei are stained in blue.
Figure Legend Snippet: KLF5 and Ki67 co-localize to the isthmal region. KLF5 and Ki67 immunohistochemistry staining was assessed on murine gastric tissue sections from uninfected mice (A and C) or H. pylori PMSS1-infected mice (B and D) at 400× magnification. Insets demonstrate regions of KLF5 and Ki67 co-localization (arrows) within the isthmal regions of the gastric epithelium (E and F). Nuclei are stained in blue.

Techniques Used: Immunohistochemistry, Staining, Mouse Assay, Infection

H. pylori -induced KLF5 upregulation is independent of the cag pathogenicity island, VacA, or LPS. AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190, or its isogenic cagE − , cagA − , slt − , or vacA − mutants at an MOI of 100∶1 for 2 hours. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were co-cultured with the wild-type cag + H. pylori strain 60190, heat-killed (HK) H. pylori strain 60190, or with strain 60190 in a transwell (TW) system for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (E) Gastric epithelial cells were treated with H. pylori LPS (10 ng/ml or 100 ng/ml) for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. Data are represented as fold over uninfected (UI) control. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.
Figure Legend Snippet: H. pylori -induced KLF5 upregulation is independent of the cag pathogenicity island, VacA, or LPS. AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190, or its isogenic cagE − , cagA − , slt − , or vacA − mutants at an MOI of 100∶1 for 2 hours. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were co-cultured with the wild-type cag + H. pylori strain 60190, heat-killed (HK) H. pylori strain 60190, or with strain 60190 in a transwell (TW) system for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (E) Gastric epithelial cells were treated with H. pylori LPS (10 ng/ml or 100 ng/ml) for 2 hours and quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. Data are represented as fold over uninfected (UI) control. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.

Techniques Used: Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY

H. pylori upregulates KLF5 in human gastric epithelial cells in vitro . AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190 at an MOI of 100∶1 for the indicated time points. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were either left untreated or pretreated with actinomycin D for 1 hour prior to co-culture with H. pylori . Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. Data are represented as fold over uninfected control. − and + symbols indicate the absence or presence of H. pylori ( Hp ), respectively. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.
Figure Legend Snippet: H. pylori upregulates KLF5 in human gastric epithelial cells in vitro . AGS human gastric epithelial cells were co-cultured with wild-type cag + H. pylori strain 60190 at an MOI of 100∶1 for the indicated time points. (A) Quantitative real-time RT-PCR was used to assess KLF5 mRNA expression relative to GAPDH mRNA expression. (B) Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. (C) Western blot analysis replicates were quantified using densitometry. (D) Gastric epithelial cells were either left untreated or pretreated with actinomycin D for 1 hour prior to co-culture with H. pylori . Western blot analysis was used to assess KLF5 protein expression relative to GAPDH protein expression. Data are represented as fold over uninfected control. − and + symbols indicate the absence or presence of H. pylori ( Hp ), respectively. Error bars indicate standard error of the mean from experiments performed on at least three independent occasions, and Mann-Whitney tests were used to determine statistical significance between groups.

Techniques Used: In Vitro, Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Co-Culture Assay, MANN-WHITNEY

H. pylori induces expansion of a KLF5 + , Lrig1 + cell population in vivo . (A) Flow cytometry dot plots demonstrate Lrig1 and KLF5 immunostaining in representative gastric epithelial cells from uninfected and H. pylori -infected mice at 4 and 8 weeks. The percentage of Lrig1 + , KLF5 + cells was quantified in uninfected and H. pylori -infected mice at 4 weeks (B) and 8 weeks (C). Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
Figure Legend Snippet: H. pylori induces expansion of a KLF5 + , Lrig1 + cell population in vivo . (A) Flow cytometry dot plots demonstrate Lrig1 and KLF5 immunostaining in representative gastric epithelial cells from uninfected and H. pylori -infected mice at 4 and 8 weeks. The percentage of Lrig1 + , KLF5 + cells was quantified in uninfected and H. pylori -infected mice at 4 weeks (B) and 8 weeks (C). Each data point represents gastric epithelial cells analyzed from a single animal and mean values are shown. Circles designate uninfected mice, and squares represent H. pylori -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

Techniques Used: In Vivo, Flow Cytometry, Cytometry, Immunostaining, Infection, Mouse Assay, MANN-WHITNEY

KLF5 expression parallels the severity of gastric premalignant lesions in H. pylori -infected humans. (A) KLF5 expression was evaluated by immunohistochemistry in a human population at high risk for gastric cancer. Gastric biopsies from uninfected patients with normal gastric mucosa and H. pylori -infected patients with non-atrophic gastritis, intestinal metaplasia (IM), and dysplasia were evaluated for KLF5 immunostaining at 200× magnification. (B and C) A single pathologist assessed the percentage of KLF5 + cells exhibiting cytoplasmic (B) or nuclear (C) staining. Each data point represents an individual biopsy and mean values are shown. The percentage and mean value of KLF5 + cells from biopsies from patients with normal gastric tissue (circles), gastritis (squares), intestinal metaplasia (IM, triangles), and dysplasia (inverted triangles) are shown. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
Figure Legend Snippet: KLF5 expression parallels the severity of gastric premalignant lesions in H. pylori -infected humans. (A) KLF5 expression was evaluated by immunohistochemistry in a human population at high risk for gastric cancer. Gastric biopsies from uninfected patients with normal gastric mucosa and H. pylori -infected patients with non-atrophic gastritis, intestinal metaplasia (IM), and dysplasia were evaluated for KLF5 immunostaining at 200× magnification. (B and C) A single pathologist assessed the percentage of KLF5 + cells exhibiting cytoplasmic (B) or nuclear (C) staining. Each data point represents an individual biopsy and mean values are shown. The percentage and mean value of KLF5 + cells from biopsies from patients with normal gastric tissue (circles), gastritis (squares), intestinal metaplasia (IM, triangles), and dysplasia (inverted triangles) are shown. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

Techniques Used: Expressing, Infection, Immunohistochemistry, Immunostaining, Staining, MANN-WHITNEY

H. pylori upregulates KLF5 expression in vivo . (A–C) KLF5 expression in murine antral gastric tissue was assessed by KLF5 immunostaining in uninfected (A), H. pylori PMSS1-infected mice (B), and H. pylori PMSS1 cagE − -infected mice (C) at 400× magnification. (D and E) A single pathologist, blinded to treatment groups, assessed and scored KLF5 immunostaining. KLF5 immunohistochemistry (IHC) score was determined by assessing the percentage of KLF5 + epithelial cells multiplied by the intensity of epithelial KLF5 staining (1–3) in both the cytoplasm and nucleus of murine gastric epithelial cells (D and E). Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE − -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.
Figure Legend Snippet: H. pylori upregulates KLF5 expression in vivo . (A–C) KLF5 expression in murine antral gastric tissue was assessed by KLF5 immunostaining in uninfected (A), H. pylori PMSS1-infected mice (B), and H. pylori PMSS1 cagE − -infected mice (C) at 400× magnification. (D and E) A single pathologist, blinded to treatment groups, assessed and scored KLF5 immunostaining. KLF5 immunohistochemistry (IHC) score was determined by assessing the percentage of KLF5 + epithelial cells multiplied by the intensity of epithelial KLF5 staining (1–3) in both the cytoplasm and nucleus of murine gastric epithelial cells (D and E). Each data point represents an individual animal and mean values are shown. Circles designate uninfected mice, squares represent H. pylori PMSS1-infected mice, and triangles represent H. pylori PMSS1 cagE − -infected mice. Mann-Whitney and ANOVA tests were used to determine statistical significance between groups.

Techniques Used: Expressing, In Vivo, Immunostaining, Infection, Mouse Assay, Immunohistochemistry, Staining, MANN-WHITNEY

5) Product Images from "Pervasive Transcription of a Herpesvirus Genome Generates Functionally Important RNAs"

Article Title: Pervasive Transcription of a Herpesvirus Genome Generates Functionally Important RNAs

Journal: mBio

doi: 10.1128/mBio.01033-13

EGR 26c ASO decreases specific genes of all kinetic classes. 3T12 cells transfected with GFP or EGR 26c ASO or untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A, C, and E) or transcript levels (B, D, and F). (A) Representative Western blots for M9 and actin at 18 hpi (2 experiments). (B) Representative Northern blot using a probe to M3 or actin at 14 hpi and corresponding quantification of M3 transcript levels normalized to those of actin (means and SEMs from 3 experiments). A 0.5-µg portion of RNA was used per lane for M3 Northern blots. (C) Representative Western blot for M3 protein at 18 hpi and corresponding quantification of M3 protein levels normalized to those of actin (means and SEMs from 4 experiments). (D) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin for cells transfected with GFP or EGR 26 ASOs (means and SEMs from 3 to 5 experiments). (E) Representative Western blot for ORF 6 and actin at 18 hpi and corresponding quantification of ORF 6 protein normalized to actin (means and SEMs from 5 experiments). Representative Western blots for ORF 6 are the same as in Fig. 1 . (F) ORF 50 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed, and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 50 transcripts or GAPDH. Data are relative ORF 50 abundances normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 4 experiments). Statistical analyses were performed by paired t test (A, B, C, and E) or one-way ANOVA with Dunnett’s posttest (D and F). *, P
Figure Legend Snippet: EGR 26c ASO decreases specific genes of all kinetic classes. 3T12 cells transfected with GFP or EGR 26c ASO or untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A, C, and E) or transcript levels (B, D, and F). (A) Representative Western blots for M9 and actin at 18 hpi (2 experiments). (B) Representative Northern blot using a probe to M3 or actin at 14 hpi and corresponding quantification of M3 transcript levels normalized to those of actin (means and SEMs from 3 experiments). A 0.5-µg portion of RNA was used per lane for M3 Northern blots. (C) Representative Western blot for M3 protein at 18 hpi and corresponding quantification of M3 protein levels normalized to those of actin (means and SEMs from 4 experiments). (D) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin for cells transfected with GFP or EGR 26 ASOs (means and SEMs from 3 to 5 experiments). (E) Representative Western blot for ORF 6 and actin at 18 hpi and corresponding quantification of ORF 6 protein normalized to actin (means and SEMs from 5 experiments). Representative Western blots for ORF 6 are the same as in Fig. 1 . (F) ORF 50 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed, and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 50 transcripts or GAPDH. Data are relative ORF 50 abundances normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 4 experiments). Statistical analyses were performed by paired t test (A, B, C, and E) or one-way ANOVA with Dunnett’s posttest (D and F). *, P

Techniques Used: Allele-specific Oligonucleotide, Transfection, Infection, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction

Effect on immediate-early, early, and late genes by EGR 27. 3T12 cells transfected with GFP or ASOs targeting EGR 27 or left untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A and D) or transcript levels (B, C, and E). See Fig. 5 and also Table S1 in the supplemental material for ASO locations. (A) Representative Western blots for M9 and ORF 26 proteins at 18 hpi (2 or 3 experiments). (B) ORF 29 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed (RT), and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 29 transcripts or GAPDH. Data are relative ORF 29 abundance normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 3 to 8 experiments). (C) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin and compared to the value for untransfected cells (means and SEMs from 5 to 7 experiments). (D) Representative Western blot for ORF 6 and actin at 18 hpi (3 experiments). The representative experiment is the one whose results are shown in panel A. (E) ORF 50 transcript levels at 14 hpi measured by qRT-PCR, as for panel B. Statistical analyses were performed by one-way ANOVA with Dunnett’s posttest. *, P
Figure Legend Snippet: Effect on immediate-early, early, and late genes by EGR 27. 3T12 cells transfected with GFP or ASOs targeting EGR 27 or left untransfected (No ASO) were infected with MHV68 (MOI = 10) and analyzed for protein (A and D) or transcript levels (B, C, and E). See Fig. 5 and also Table S1 in the supplemental material for ASO locations. (A) Representative Western blots for M9 and ORF 26 proteins at 18 hpi (2 or 3 experiments). (B) ORF 29 transcript levels at 14 hpi. RNA (1 µg) was reverse transcribed (RT), and cDNA was analyzed by qPCR using primers designed to detect spliced ORF 29 transcripts or GAPDH. Data are relative ORF 29 abundance normalized to GAPDH transcript abundance and compared to untransfected cells by the ∆∆ C T method (means and SEMs from 3 to 8 experiments). (C) Representative Northern blot for ORF 6 and actin at 14 hpi and corresponding quantification of ORF 6 transcript levels normalized to actin and compared to the value for untransfected cells (means and SEMs from 5 to 7 experiments). (D) Representative Western blot for ORF 6 and actin at 18 hpi (3 experiments). The representative experiment is the one whose results are shown in panel A. (E) ORF 50 transcript levels at 14 hpi measured by qRT-PCR, as for panel B. Statistical analyses were performed by one-way ANOVA with Dunnett’s posttest. *, P

Techniques Used: Transfection, Allele-specific Oligonucleotide, Infection, Western Blot, Real-time Polymerase Chain Reaction, Northern Blot, Quantitative RT-PCR

An antisense oligonucleotide to ORF 6 decreases ORF 6 transcript and protein expression and late-gene expression. 3T12 cells transfected with ASOs targeting ORF 6, M3, or GFP (negative control) or left untransfected (No ASO) were infected with MHV68. (A) Representative Northern blot for ORF 6 or actin transcripts at 14 hpi and corresponding quantification of ORF 6 monocistronic transcript levels normalized to those of actin (MOI = 10; values are means and standard errors of the means [SEMs] from 3 experiments; **, P
Figure Legend Snippet: An antisense oligonucleotide to ORF 6 decreases ORF 6 transcript and protein expression and late-gene expression. 3T12 cells transfected with ASOs targeting ORF 6, M3, or GFP (negative control) or left untransfected (No ASO) were infected with MHV68. (A) Representative Northern blot for ORF 6 or actin transcripts at 14 hpi and corresponding quantification of ORF 6 monocistronic transcript levels normalized to those of actin (MOI = 10; values are means and standard errors of the means [SEMs] from 3 experiments; **, P

Techniques Used: Expressing, Transfection, Negative Control, Allele-specific Oligonucleotide, Infection, Northern Blot

6) Product Images from "Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression"

Article Title: Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression

Journal: Scientific Reports

doi: 10.1038/srep19781

PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P
Figure Legend Snippet: PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P

Techniques Used: Mouse Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

7) Product Images from "Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression"

Article Title: Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression

Journal: Scientific Reports

doi: 10.1038/srep19781

PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P
Figure Legend Snippet: PGC1 α1 levels increase in the soleus in trained mice and in transfected C2C12 cells upon transfection with pCMV-Entry-HSPD1 vector. ( A ) representative western blots of soleus and relative expression levels (bars) of PGC1 α1 (113 kDa), 4 HNE (55 kDa), Mn SOD (25 kDa), p-AMPKα (63 kDa), AMPKα1 (63 kDa), AMPKα2 (63 kDa), TFAM (30 kDa) in soleus of sedentary (SED45, open bar, n = 8) and trained (TR45, shaded bar, n = 8) mice at 45 days. 80 μg of proteins were loaded in each lane; GAPDH (37 kDa) was used as the loading control. Data are presented as the means ± SD. ∂ significantly different from TR45 mice (P

Techniques Used: Mouse Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

8) Product Images from "Extracorporeal immune therapy with immobilized agonistic anti-Fas antibodies leads to transient reduction of circulating neutrophil numbers and limits tissue damage after hemorrhagic shock/resuscitation in a porcine model"

Article Title: Extracorporeal immune therapy with immobilized agonistic anti-Fas antibodies leads to transient reduction of circulating neutrophil numbers and limits tissue damage after hemorrhagic shock/resuscitation in a porcine model

Journal: Journal of Inflammation (London, England)

doi: 10.1186/1476-9255-7-18

Heme oxygenase-1 (HO-1) gene expression (A), and HO-1 protein expression (B) in control (white bars), SMC (grey bars), and LIM (black bars) animals .
Figure Legend Snippet: Heme oxygenase-1 (HO-1) gene expression (A), and HO-1 protein expression (B) in control (white bars), SMC (grey bars), and LIM (black bars) animals .

Techniques Used: Expressing

9) Product Images from "Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion"

Article Title: Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

Journal: mBio

doi: 10.1128/mBio.01802-14

(A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.
Figure Legend Snippet: (A) Fibers containing SEPT2, -9, and -11 encase the chlamydial inclusion. HeLa cells infected for 48 h were fixed and stained for SEPT2 or SEPT9 or infected for 30 h, fixed, and stained for SEPT11. Chlamydial inclusions are indicated by arrows. Cell nuclei are marked with asterisks. Scale bar, 10 µm. Images are representative of at least 3 independent experiments. Proteomic analysis found the four septins SEPT2, -7, -9, and -11 on purified inclusions (see Materials and Methods for details; the following numbers of peptides were identified by mass spectrometry in infected/uninfected cells: SEPT2, 64/29; SEPT7, 6/11; SEPT9, 13/5; SEPT11, 7/11). (B) Infection increases the amount of SEPT9 in SEPT2-containing complexes. Uninfected HeLa cells or HeLa cells infected with C. trachomatis for 30 h were lysed, and proteins in the lysate supernatants (Input) were immunoprecipitated with anti-SEPT2 antibodies. Input, unbound, and IP fractions from the immunoprecipitations (IPs) were analyzed by SDS-PAGE, followed by immunoblotting with anti-SEPT2, -7, -9, or -11 or anti-GAPDH antibody. The smaller band for SEPT2 is consistent with cleavage by CPAF during sample preparation. A number of isoforms exist for SEPT9; one isoform especially appears to be recruited to septin fibers during infection.

Techniques Used: Infection, Staining, Purification, Mass Spectrometry, Immunoprecipitation, SDS Page, Sample Prep

10) Product Images from "Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor"

Article Title: Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/278273

Isorhamnetin potentiates the NGF-induced neurofilament expression. Cultured PC12 cells were treated with NGF (0.5 ng/mL), isorhamnetin (10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (10 μ M) for 72 hours. NGF at 50 ng/mL was applied as a control. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. Quantification plot was shown in lower panel. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. ** where P
Figure Legend Snippet: Isorhamnetin potentiates the NGF-induced neurofilament expression. Cultured PC12 cells were treated with NGF (0.5 ng/mL), isorhamnetin (10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (10 μ M) for 72 hours. NGF at 50 ng/mL was applied as a control. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. Quantification plot was shown in lower panel. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. ** where P

Techniques Used: Expressing, Cell Culture

Isorhamnetin induces the neurofilament expression in cultured PC12 cells but not the neurite outgrowth. (a) The chemical structure of isorhamnetin is illustrated. (b) Cultured PC12 cells were treated with isorhamnetin (1 to10 μ M) for 72 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. The lower panel shows the quantitation from the blots by a densitometer. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4, each with triplicate samples. (c) Cultures were treated with isorhamnetin (3 or 10 μ M) and NGF (50 ng/mL), as indicated, for 72 hours. Cells were fixed with ice-cold 4% paraformaldehyde. Bar = 10 μ m. Representative images were shown. (d) Cultured PC12 cell was treated as in (c). The % of differentiated cell (upper panel) and length of neurite (lower panel) were counted as described in the Materials and Methods section. Values are expressed as % of total cells in 100 counted cells, mean ± SEM, n = 4. ** P
Figure Legend Snippet: Isorhamnetin induces the neurofilament expression in cultured PC12 cells but not the neurite outgrowth. (a) The chemical structure of isorhamnetin is illustrated. (b) Cultured PC12 cells were treated with isorhamnetin (1 to10 μ M) for 72 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. The lower panel shows the quantitation from the blots by a densitometer. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4, each with triplicate samples. (c) Cultures were treated with isorhamnetin (3 or 10 μ M) and NGF (50 ng/mL), as indicated, for 72 hours. Cells were fixed with ice-cold 4% paraformaldehyde. Bar = 10 μ m. Representative images were shown. (d) Cultured PC12 cell was treated as in (c). The % of differentiated cell (upper panel) and length of neurite (lower panel) were counted as described in the Materials and Methods section. Values are expressed as % of total cells in 100 counted cells, mean ± SEM, n = 4. ** P

Techniques Used: Expressing, Cell Culture, Quantitation Assay

The potentiating effect of isorhamnetin on NGF-induced response could not be blocked by U0126. Cultured PC12 cells, serum starvation for 5 hours, were treated with NGF (0.5 ng/mL), isorhamnetin (Iso; 10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (Iso; 10 μ M) for 72 hours with or without the pretreatment of U0126 (20 μ M) for 3 hours. NGF at 50 ng/mL served as a positive control. (a) The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. GADPH served as a loading control. (b) Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. **where P
Figure Legend Snippet: The potentiating effect of isorhamnetin on NGF-induced response could not be blocked by U0126. Cultured PC12 cells, serum starvation for 5 hours, were treated with NGF (0.5 ng/mL), isorhamnetin (Iso; 10 μ M), and NGF (0.5 ng/mL) + isorhamnetin (Iso; 10 μ M) for 72 hours with or without the pretreatment of U0126 (20 μ M) for 3 hours. NGF at 50 ng/mL served as a positive control. (a) The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. GADPH served as a loading control. (b) Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. **where P

Techniques Used: Cell Culture, Positive Control

NGF induces the expression of neurofilaments in cultured PC12 cells. Cultured PC12 cells were treated with NGF (0.3 to 50 ng/mL) for 72 hours. The cell lysates were collected to determine the expressions of NF68 ( M r ~ 68 kDa), NF160 ( M r ~ 160 kDa), and NF200 ( M r ~ 200 kDa). GADPH ( M r ~ 38 kDa) served as a loading control (upper panel). Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in Mean ± SEM, n = 4, each with triplicate samples. Representative images were shown. ** P
Figure Legend Snippet: NGF induces the expression of neurofilaments in cultured PC12 cells. Cultured PC12 cells were treated with NGF (0.3 to 50 ng/mL) for 72 hours. The cell lysates were collected to determine the expressions of NF68 ( M r ~ 68 kDa), NF160 ( M r ~ 160 kDa), and NF200 ( M r ~ 200 kDa). GADPH ( M r ~ 38 kDa) served as a loading control (upper panel). Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in Mean ± SEM, n = 4, each with triplicate samples. Representative images were shown. ** P

Techniques Used: Expressing, Cell Culture

11) Product Images from "Def-6, a Novel Regulator of Small GTPases in Podocytes, Acts Downstream of Atypical Protein Kinase C (aPKC) λ/ι"

Article Title: Def-6, a Novel Regulator of Small GTPases in Podocytes, Acts Downstream of Atypical Protein Kinase C (aPKC) λ/ι

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.08.026

Def-6 expression is enhanced in PKCλ/ι −/− glomeruli and localizes to cellular edges in podocytes. A–C : Staining of kidney cryosections from PKCλ/ι +/+ and PKCλ/ι −/− mice. A: Upper panels : Overview staining with an antibody against Def-6 (×10 magnification) reveals strong glomerular expression. Lower panels : Control staining with secondary antibody or Def-6 blocking peptide validated the specificity of the Def-6 antibody. Scale bars = 200 μm. B : Co-staining of Def-6 (red) and the podocyte marker podocalyxin (green) reveals enhanced podocytic expression of Def-6 in PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Nuclei are visualized with DAPI (blue). Scale bars = 25 μm (applies to all panels). C : For quantification of Def-6 expression, sections were double-stained against Def-6 (red) and nidogen (green). Nuclei were visualized using DAPI (blue). Def-6 expression is enhanced in the podocytic areas of PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Scale bars = 25 μm (applies to all panels). D : Images of PKCλ/ι +/+ and PKCλ/ι − / − glomeruli (40 glomeruli from four animals of each genotype) were obtained and analyzed using a semiquantitative score ranging from 0 (no expression) to 4 (strong expression). Compared with WT glomeruli, PKCλ/ι − / − glomeruli show a significantly higher score and an obvious change in score distribution. n = 4 mice of each genotype, ≥10 glomeruli per mouse. ∗∗∗ P
Figure Legend Snippet: Def-6 expression is enhanced in PKCλ/ι −/− glomeruli and localizes to cellular edges in podocytes. A–C : Staining of kidney cryosections from PKCλ/ι +/+ and PKCλ/ι −/− mice. A: Upper panels : Overview staining with an antibody against Def-6 (×10 magnification) reveals strong glomerular expression. Lower panels : Control staining with secondary antibody or Def-6 blocking peptide validated the specificity of the Def-6 antibody. Scale bars = 200 μm. B : Co-staining of Def-6 (red) and the podocyte marker podocalyxin (green) reveals enhanced podocytic expression of Def-6 in PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Nuclei are visualized with DAPI (blue). Scale bars = 25 μm (applies to all panels). C : For quantification of Def-6 expression, sections were double-stained against Def-6 (red) and nidogen (green). Nuclei were visualized using DAPI (blue). Def-6 expression is enhanced in the podocytic areas of PKCλ/ι − / − glomeruli ( boxed areas in Merge views, and arrowhead s in Detail views). Scale bars = 25 μm (applies to all panels). D : Images of PKCλ/ι +/+ and PKCλ/ι − / − glomeruli (40 glomeruli from four animals of each genotype) were obtained and analyzed using a semiquantitative score ranging from 0 (no expression) to 4 (strong expression). Compared with WT glomeruli, PKCλ/ι − / − glomeruli show a significantly higher score and an obvious change in score distribution. n = 4 mice of each genotype, ≥10 glomeruli per mouse. ∗∗∗ P

Techniques Used: Expressing, Staining, Mouse Assay, Blocking Assay, Marker

12) Product Images from "Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity"

Article Title: Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity

Journal: Molecular Endocrinology

doi: 10.1210/me.2008-0211

PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG
Figure Legend Snippet: PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG

Techniques Used: Expressing, Mutagenesis, Western Blot, Over Expression

13) Product Images from "14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection"

Article Title: 14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection

Journal: Veterinary Research

doi: 10.1186/s13567-019-0636-0

Identification of interactions between 14-3-3 subtypes and NSP2 by confocal microscopy (A and C) and IP (B). A Co-localization of NSP2 with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of EGFP–NSP2 (green) with 14-3-3 β, ε, γ, and τ/ζ (red) was visualized in transfected 293T cells. B 293T cells were transfected with GFP–nsp2 and an empty vector. Proteins associated with NSP2 were pulled down using GFP-Trap and analyzed by Western blot using specific antibodies against 14-3-3β, ε, γ, and τ/ζ. C Co-localization of HP-PRRSV with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of NSP2 of TA-12 (green) with 14-3-3 α/β, ε, γ, and τ/ζ (red) was visualized in Marc-145 cells. Co-localization was determined by the yellow signal in merged images. WCL: whole-cell lysates.
Figure Legend Snippet: Identification of interactions between 14-3-3 subtypes and NSP2 by confocal microscopy (A and C) and IP (B). A Co-localization of NSP2 with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of EGFP–NSP2 (green) with 14-3-3 β, ε, γ, and τ/ζ (red) was visualized in transfected 293T cells. B 293T cells were transfected with GFP–nsp2 and an empty vector. Proteins associated with NSP2 were pulled down using GFP-Trap and analyzed by Western blot using specific antibodies against 14-3-3β, ε, γ, and τ/ζ. C Co-localization of HP-PRRSV with 14-3-3 subtypes, confirmed by immunofluorescence microscopy. Co-localization of NSP2 of TA-12 (green) with 14-3-3 α/β, ε, γ, and τ/ζ (red) was visualized in Marc-145 cells. Co-localization was determined by the yellow signal in merged images. WCL: whole-cell lysates.

Techniques Used: Confocal Microscopy, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation, Western Blot

14) Product Images from "Epigenetic reprogramming converts human Wharton’s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators"

Article Title: Epigenetic reprogramming converts human Wharton’s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0638-7

a Study of cardiac morphology after induction with different epigenetic modifiers. Differentiation was performed with DC301, DC302, and DC303, and various combinations of these inhibitors, and the appearance of cardiac morphology was assessed by phase-contrast microscopy ( scale bars = 100 μm, n = 3). Treatment with DC301 + DC302 indicates clear cardiac morphology ( scale bar = 50 μm). b Analysis of cardiac markers by quantitative RT-PCR after cardiac induction with different epigenetic modifiers. Cardiac-specific genes GATA4 , Nkx2.5 , MLC , TnT , and cardiac actin were studied for their expression after cardiac induction with DC301, DC302, and DC303, and various combinations of these inhibitors as indicated. Results are mean ± SD of three independent experiments performed in triplicate (* p
Figure Legend Snippet: a Study of cardiac morphology after induction with different epigenetic modifiers. Differentiation was performed with DC301, DC302, and DC303, and various combinations of these inhibitors, and the appearance of cardiac morphology was assessed by phase-contrast microscopy ( scale bars = 100 μm, n = 3). Treatment with DC301 + DC302 indicates clear cardiac morphology ( scale bar = 50 μm). b Analysis of cardiac markers by quantitative RT-PCR after cardiac induction with different epigenetic modifiers. Cardiac-specific genes GATA4 , Nkx2.5 , MLC , TnT , and cardiac actin were studied for their expression after cardiac induction with DC301, DC302, and DC303, and various combinations of these inhibitors as indicated. Results are mean ± SD of three independent experiments performed in triplicate (* p

Techniques Used: Microscopy, Quantitative RT-PCR, Expressing

Nkx2.5 promoter CpG island demethylation. a Position of Nkx2.5 gene on chromosome 5, sequence of the promoter region indicated. b After bisulfite conversion, the 160-bp promoter region of Nkx2.5 was amplified and cloned for bisulfite sequencing for control ( U ) and differentiated cardiomyocytes ( D ); shaded regions indicate the CpG islands that are modified after cardiac differentiation. c CpG islands were evaluated as methylated ( orange dot ) and unmethylated ( red dot ) sites by bisulfite sequencing. d After bisulfite conversion, methylation-specific PCR was performed for Nkx2.5 using unmethylated and methylated primers in control (U) and differentiated cardiomyocytes (D) (** p
Figure Legend Snippet: Nkx2.5 promoter CpG island demethylation. a Position of Nkx2.5 gene on chromosome 5, sequence of the promoter region indicated. b After bisulfite conversion, the 160-bp promoter region of Nkx2.5 was amplified and cloned for bisulfite sequencing for control ( U ) and differentiated cardiomyocytes ( D ); shaded regions indicate the CpG islands that are modified after cardiac differentiation. c CpG islands were evaluated as methylated ( orange dot ) and unmethylated ( red dot ) sites by bisulfite sequencing. d After bisulfite conversion, methylation-specific PCR was performed for Nkx2.5 using unmethylated and methylated primers in control (U) and differentiated cardiomyocytes (D) (** p

Techniques Used: Sequencing, Amplification, Clone Assay, Methylation Sequencing, Modification, Methylation, Polymerase Chain Reaction

15) Product Images from "Calcineurin in Reactive Astrocytes Plays a Key Role in the Interplay between Proinflammatory and Anti-Inflammatory Signals"

Article Title: Calcineurin in Reactive Astrocytes Plays a Key Role in the Interplay between Proinflammatory and Anti-Inflammatory Signals

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1002-07.2007

Astrocytic calcineurin and neuroinflammatory damage. A , Astrocytes transduced with ΔCnA show increased activity of calcineurin (measured as release of PO 4 ) compared with mock-transfected (CMV) or wt astrocytes (histograms). Only ΔCnA-transfected astrocytes produced the truncated mutant form of calcineurin (ΔCnA; blots). Levels of endogenous calcineurin remained unaffected. B , Schedule followed for coculture of astrocytes and neurons. Astrocytes were plated and transfected with corresponding DNAs, and neurons were added 24 h later. Thereafter, cocultures were challenged with inflammatory stimuli (LPS or TNF-α) for various times before analysis of neuronal death after a total of 4 d in coculture. Inhibitors such as CsA or MG-132 were added at indicated times. C , Photomicrographs, Representative double immunocytochemical staining used to identify apoptotic (activated caspase 3 + cells; red) neurons (β3-tubulin + cells; green) after inflammatory challenge. Histograms, Neurons cocultured with mock-transfected astrocytes (CMV; striped left histograms) die soon after LPS (top histograms) or TNF-α addition (bottom histograms), whereas when cocultured with astrocytes expressing ΔCnA (black right histograms), they show a significantly greater resistance to these inflammatory stimuli (** p
Figure Legend Snippet: Astrocytic calcineurin and neuroinflammatory damage. A , Astrocytes transduced with ΔCnA show increased activity of calcineurin (measured as release of PO 4 ) compared with mock-transfected (CMV) or wt astrocytes (histograms). Only ΔCnA-transfected astrocytes produced the truncated mutant form of calcineurin (ΔCnA; blots). Levels of endogenous calcineurin remained unaffected. B , Schedule followed for coculture of astrocytes and neurons. Astrocytes were plated and transfected with corresponding DNAs, and neurons were added 24 h later. Thereafter, cocultures were challenged with inflammatory stimuli (LPS or TNF-α) for various times before analysis of neuronal death after a total of 4 d in coculture. Inhibitors such as CsA or MG-132 were added at indicated times. C , Photomicrographs, Representative double immunocytochemical staining used to identify apoptotic (activated caspase 3 + cells; red) neurons (β3-tubulin + cells; green) after inflammatory challenge. Histograms, Neurons cocultured with mock-transfected astrocytes (CMV; striped left histograms) die soon after LPS (top histograms) or TNF-α addition (bottom histograms), whereas when cocultured with astrocytes expressing ΔCnA (black right histograms), they show a significantly greater resistance to these inflammatory stimuli (** p

Techniques Used: Transduction, Activity Assay, Transfection, Produced, Mutagenesis, Staining, Expressing

Astrocytic calcineurin protects against LPS-induced inflammatory damage. A , Three days after intraparenchymael injection of LPS, reactive astrocytes (GFAP + ) in AIC mice not treated with Dox show negligible iNOS2 immunoreactivity. Scale bar, 100 μm. B , This was paralleled by a drastic reduction in levels of Cox2 and iNOS2 in the injected area ( p
Figure Legend Snippet: Astrocytic calcineurin protects against LPS-induced inflammatory damage. A , Three days after intraparenchymael injection of LPS, reactive astrocytes (GFAP + ) in AIC mice not treated with Dox show negligible iNOS2 immunoreactivity. Scale bar, 100 μm. B , This was paralleled by a drastic reduction in levels of Cox2 and iNOS2 in the injected area ( p

Techniques Used: Injection, Mouse Assay

Calcineurin inhibits the NFκB/NFAT proinflammatory pathway in astrocytes. A , LPS failed to stimulate Cox2 and iNOS2 in astrocytes expressing ΔCnA. A representative blot is shown ( n = 6). B , LPS- or TNF-α-induced activation of NFκB and NFAT was abrogated in ΔCnA-transduced astrocytes. The activity of these transcription factors was inhibited after exposure to LPS/TNF-α in ΔCnA-expressing astrocytes. Note that expression of ΔCnA in unstimulated astrocytes reduced the activity of NFκB and NFAT. CMV, Astrocytes transfected with the empty vector; ΔCnA, astrocytes transfected with constitutively active calcineurin; CMV+NFκB or NFAT, mock-transfected astrocytes expressing the gene-reporter system for either transcription factor; ΔCnA+NFκB or NFAT, ΔCnA-transfected astrocytes expressing the gene-reporter system. *** p
Figure Legend Snippet: Calcineurin inhibits the NFκB/NFAT proinflammatory pathway in astrocytes. A , LPS failed to stimulate Cox2 and iNOS2 in astrocytes expressing ΔCnA. A representative blot is shown ( n = 6). B , LPS- or TNF-α-induced activation of NFκB and NFAT was abrogated in ΔCnA-transduced astrocytes. The activity of these transcription factors was inhibited after exposure to LPS/TNF-α in ΔCnA-expressing astrocytes. Note that expression of ΔCnA in unstimulated astrocytes reduced the activity of NFκB and NFAT. CMV, Astrocytes transfected with the empty vector; ΔCnA, astrocytes transfected with constitutively active calcineurin; CMV+NFκB or NFAT, mock-transfected astrocytes expressing the gene-reporter system for either transcription factor; ΔCnA+NFκB or NFAT, ΔCnA-transfected astrocytes expressing the gene-reporter system. *** p

Techniques Used: Expressing, Activation Assay, Activity Assay, Transfection, Plasmid Preparation

Regulated expression of ΔCnA in astrocytes. A , Coexpression of GFAP–tTA (GFAP) and TetO–ΔCnA (ΔCnA) in cultured wt astrocytes allows Dox-regulated activation of calcineurin. Both control (wt) and GFAP–tTA-transduced astrocytes show lower and Dox-independent calcineurin activity. *** p
Figure Legend Snippet: Regulated expression of ΔCnA in astrocytes. A , Coexpression of GFAP–tTA (GFAP) and TetO–ΔCnA (ΔCnA) in cultured wt astrocytes allows Dox-regulated activation of calcineurin. Both control (wt) and GFAP–tTA-transduced astrocytes show lower and Dox-independent calcineurin activity. *** p

Techniques Used: Expressing, Cell Culture, Activation Assay, Activity Assay

Stages of the neuroinflammatory process in which astrocyte calcineurin may participate. Initiation, Inflammatory signals set in motion by the neuropathological process activate calcineurin, which in turn activate the canonical NFκB/NFAT pathway. Activation of local and peripheral proinflammatory mechanisms together with the recruitment of autocrine and paracrine neuroprotective mediators follows. The time course of this simultaneous anti-inflammatory and proinflammatory cascade may be critical to the eventual outcome of the inflammatory response. Both agonistic and antagonistic inflammatory signals are produced by reactive astrocytes and microglia, damaged neurons and activated endothelia, and eventually from peripheral cells recruited to the lesion site. Resolution, If already activated calcineurin is stimulated by signals such as IGF-I, a neuroprotective network is activated; Progression, if calcineurin continues to be activated by inflammatory signals, the inflammation proceeds and neurons die. Both phases may be reversibly interrelated depending on the time course of the pathological process. Mechanisms whereby calcineurin is recruited toward either inflammation or neuroprotection, which involve differential interactions with transcription factors such as PPARγ and GATA3 or proteasome degradation and which depend on the upstream signal stimulating calcineurin, warrant additional analysis.
Figure Legend Snippet: Stages of the neuroinflammatory process in which astrocyte calcineurin may participate. Initiation, Inflammatory signals set in motion by the neuropathological process activate calcineurin, which in turn activate the canonical NFκB/NFAT pathway. Activation of local and peripheral proinflammatory mechanisms together with the recruitment of autocrine and paracrine neuroprotective mediators follows. The time course of this simultaneous anti-inflammatory and proinflammatory cascade may be critical to the eventual outcome of the inflammatory response. Both agonistic and antagonistic inflammatory signals are produced by reactive astrocytes and microglia, damaged neurons and activated endothelia, and eventually from peripheral cells recruited to the lesion site. Resolution, If already activated calcineurin is stimulated by signals such as IGF-I, a neuroprotective network is activated; Progression, if calcineurin continues to be activated by inflammatory signals, the inflammation proceeds and neurons die. Both phases may be reversibly interrelated depending on the time course of the pathological process. Mechanisms whereby calcineurin is recruited toward either inflammation or neuroprotection, which involve differential interactions with transcription factors such as PPARγ and GATA3 or proteasome degradation and which depend on the upstream signal stimulating calcineurin, warrant additional analysis.

Techniques Used: Activation Assay, Produced

Astrocytic calcineurin participates in proinflammatory and in anti-inflammatory signaling. A , In wt astrocytes, calcineurin activity was incremented by proinflammatory stimuli such as LPS/TNF-α and by neuroprotective signals such as IGF-I. ** p
Figure Legend Snippet: Astrocytic calcineurin participates in proinflammatory and in anti-inflammatory signaling. A , In wt astrocytes, calcineurin activity was incremented by proinflammatory stimuli such as LPS/TNF-α and by neuroprotective signals such as IGF-I. ** p

Techniques Used: Activity Assay

16) Product Images from "Neuronal Wiskott–Aldrich syndrome protein regulates TGF-β1–mediated lung vascular permeability"

Article Title: Neuronal Wiskott–Aldrich syndrome protein regulates TGF-β1–mediated lung vascular permeability

Journal: The FASEB Journal

doi: 10.1096/fj.201600102R

Activation of Rho and FAK is necessary for Y256 phosphorylation of N-WASP induced by TGF-β1 in RMVECs. A ) Proposed mechanism of N-WASP activation by TGF-β1. Previous data indicate that Rho GTPase activation unlocks N-WASP from an autoinhibitory,
Figure Legend Snippet: Activation of Rho and FAK is necessary for Y256 phosphorylation of N-WASP induced by TGF-β1 in RMVECs. A ) Proposed mechanism of N-WASP activation by TGF-β1. Previous data indicate that Rho GTPase activation unlocks N-WASP from an autoinhibitory,

Techniques Used: Activation Assay

TGF-β1 induces phosphorylation of Y256 of N-WASP. N-WASP Y256 plays a critical role in TGF-β1–induced paracellular permeability and actin stress fiber formation in RMVECs. A ) TGF-β1 induces phosphorylation of Y256 of N-WASP
Figure Legend Snippet: TGF-β1 induces phosphorylation of Y256 of N-WASP. N-WASP Y256 plays a critical role in TGF-β1–induced paracellular permeability and actin stress fiber formation in RMVECs. A ) TGF-β1 induces phosphorylation of Y256 of N-WASP

Techniques Used: Permeability

17) Product Images from "Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor"

Article Title: Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001951

Effect of BZL on CYP3A4 and GST expression. Cells were exposed either to vehicle (C) or BZL (200 µM) for 48 h. CYP3A4 (panel A), GSTα (panel B), GSTμ (panel C), and GSTπ (panel D) levels were estimated by western blotting. Equal amounts of total protein (15 µg) were loaded in the gels. CYP3A4 or GST O.D. was related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p
Figure Legend Snippet: Effect of BZL on CYP3A4 and GST expression. Cells were exposed either to vehicle (C) or BZL (200 µM) for 48 h. CYP3A4 (panel A), GSTα (panel B), GSTμ (panel C), and GSTπ (panel D) levels were estimated by western blotting. Equal amounts of total protein (15 µg) were loaded in the gels. CYP3A4 or GST O.D. was related to β-actin O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. The data on O.D. (% of C) are presented as mean ± S.D. (n = 3). Typical western blot detections are shown at the bottom. *Significantly different from C, p

Techniques Used: Expressing, Western Blot

Effect of PXR knock down on BZL mediated P-gp, MRP2, CYP3A4 and GSTπ induction. P-gp (panel A), MRP2 (panel B), CYP3A4 (panel C) and GSTπ (panel D) levels were estimated by western blotting in lysates from HepG2 cells transfected either with 100 nM Control siRNA-A (PXR + ) or 100 nM PXR siRNA (h) (PXR − ) and exposed to BZL (200 µM, 48 h) or vehicle (C). Equal amounts of total protein (7 µg) were loaded in the gels. O.D. from each protein was related to GAPDH O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. Typical western blot detections from each group are shown at the bottom of bar graphics. The results (% of each control) are expressed as mean ± S.D. (n = 3). *Significantly different from C, p
Figure Legend Snippet: Effect of PXR knock down on BZL mediated P-gp, MRP2, CYP3A4 and GSTπ induction. P-gp (panel A), MRP2 (panel B), CYP3A4 (panel C) and GSTπ (panel D) levels were estimated by western blotting in lysates from HepG2 cells transfected either with 100 nM Control siRNA-A (PXR + ) or 100 nM PXR siRNA (h) (PXR − ) and exposed to BZL (200 µM, 48 h) or vehicle (C). Equal amounts of total protein (7 µg) were loaded in the gels. O.D. from each protein was related to GAPDH O.D. Uniformity of loading and transfer from gel to PVDF membrane was also controlled with Ponceau S. Typical western blot detections from each group are shown at the bottom of bar graphics. The results (% of each control) are expressed as mean ± S.D. (n = 3). *Significantly different from C, p

Techniques Used: Western Blot, Transfection

18) Product Images from "Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia"

Article Title: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia

Journal: Science signaling

doi: 10.1126/scisignal.aao5617

Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.
Figure Legend Snippet: Working model: Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.

Techniques Used: Inhibition, Mutagenesis

TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.
Figure Legend Snippet: TNK2 increases signaling through PTPN11/RAS/MAPK in cells overexpressing mutant PTPN11.

Techniques Used: Mutagenesis

A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.
Figure Legend Snippet: A primary patient sample containing a PTPN11 mutation demonstrates dasatinib sensitivity and over-reliance on TNK2.

Techniques Used: Mutagenesis

Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.
Figure Legend Snippet: Inhibition of TNK2 reduces signaling through PTPN11/RAS/MAPK.

Techniques Used: Inhibition

Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.
Figure Legend Snippet: Functional assays show increased transformation potential and sensitivity to TNK2 inhibition.

Techniques Used: Functional Assay, Transformation Assay, Inhibition

19) Product Images from "Molecular basis of arrhythmic substrate in ageing murine peroxisome proliferator-activated receptor γ co-activator deficient hearts modelling mitochondrial dysfunction"

Article Title: Molecular basis of arrhythmic substrate in ageing murine peroxisome proliferator-activated receptor γ co-activator deficient hearts modelling mitochondrial dysfunction

Journal: Bioscience Reports

doi: 10.1042/BSR20190403

IF analysis of atrial Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained atrial sections visualised at 40×. ( B ) Expression levels of atrial Cx43 as obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.
Figure Legend Snippet: IF analysis of atrial Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained atrial sections visualised at 40×. ( B ) Expression levels of atrial Cx43 as obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.

Techniques Used: Expressing, Staining, Mouse Assay

WB analysis of atrial Na V 1.5, Cx40 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of atrial Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of atrial Cx40 obtained by densitometric analysis. ( D ) Expression levels of atrial Cx43 expression obtained by densitometric analysis. ( E ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. In (B–D), red boxes indicate young mice and blue boxes indicate aged mice. Primary monoclonal rabbit anti-Na V 1.5 antibodies used at dilution 1:500; polyclonal goat anti-Cx40 used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used in blots staining for Cx40 at dilution 1:15000. Donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD test are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-A, wild type young atrial.
Figure Legend Snippet: WB analysis of atrial Na V 1.5, Cx40 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of atrial Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of atrial Cx40 obtained by densitometric analysis. ( D ) Expression levels of atrial Cx43 expression obtained by densitometric analysis. ( E ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. In (B–D), red boxes indicate young mice and blue boxes indicate aged mice. Primary monoclonal rabbit anti-Na V 1.5 antibodies used at dilution 1:500; polyclonal goat anti-Cx40 used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used in blots staining for Cx40 at dilution 1:15000. Donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD test are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-A, wild type young atrial.

Techniques Used: Western Blot, Expressing, Mouse Assay, Staining

WB analysis of ventricular Na V 1.5 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of ventricular Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of ventricular Cx43 obtained by densitometric analysis. In (B,C), red boxes indicate young mice and blue boxes indicate aged mice. ( D ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. Primary monoclonal rabbit anti-Na V 1.5 antibody used at dilution 1:500; polyclonal goat anti-Cx40 antibody used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used at dilution 1:15000 in blots staining for Cx40, and donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-V, wild type young ventricular.
Figure Legend Snippet: WB analysis of ventricular Na V 1.5 and Cx43 expression ( A ) Representative Western blots of Na V 1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. ( B ) Expression levels of ventricular Na V 1.5 obtained by densitometric analysis. ( C ) Expression levels of ventricular Cx43 obtained by densitometric analysis. In (B,C), red boxes indicate young mice and blue boxes indicate aged mice. ( D ) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. Primary monoclonal rabbit anti-Na V 1.5 antibody used at dilution 1:500; polyclonal goat anti-Cx40 antibody used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used at dilution 1:15000 in blots staining for Cx40, and donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for Na V 1.5, Cx43 and GAPDH. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-V, wild type young ventricular.

Techniques Used: Western Blot, Expressing, Mouse Assay, Staining

IF analysis of ventricular Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained ventricular sections visualised at 40×. ( B ) Expression levels of ventricular Cx43 obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.
Figure Legend Snippet: IF analysis of ventricular Cx43 expression ( A ) Representative micrographs of Cx43 signal in stained ventricular sections visualised at 40×. ( B ) Expression levels of ventricular Cx43 obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P -values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.

Techniques Used: Expressing, Staining, Mouse Assay

20) Product Images from "Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity"

Article Title: Estrogen-Mediated Suppression of the Gene Encoding Protein Tyrosine Phosphatase PTPRO in Human Breast Cancer: Mechanism and Role in Tamoxifen Sensitivity

Journal: Molecular Endocrinology

doi: 10.1210/me.2008-0211

PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG
Figure Legend Snippet: PTPRO expression augments tamoxifen sensitivity of breast cancer cell line. A, The WT or CS mutant of FLAG-tagged PTPRO was overexpressed in MCF-7 cells. The G418 selected pool was analyzed by Western blot analysis for PTPRO overexpression using anti-FLAG

Techniques Used: Expressing, Mutagenesis, Western Blot, Over Expression

21) Product Images from "Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression"

Article Title: Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01126-12

2-DG but not 2-FDG induces ER stress and a UPR in lytically infected cells. (A) 293rKSHV cells were lytically induced with butyrate (3 mM) in the presence of 2-DG (1 mM) or 2-FDG (1 mM). At the indicated times postinduction, cells were harvested and immunoblotting
Figure Legend Snippet: 2-DG but not 2-FDG induces ER stress and a UPR in lytically infected cells. (A) 293rKSHV cells were lytically induced with butyrate (3 mM) in the presence of 2-DG (1 mM) or 2-FDG (1 mM). At the indicated times postinduction, cells were harvested and immunoblotting

Techniques Used: Infection

22) Product Images from "Thrombin-dependent MMP-2 Activity Is Regulated by Heparan Sulfate *"

Article Title: Thrombin-dependent MMP-2 Activity Is Regulated by Heparan Sulfate *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.171595

Heparan sulfate proteoglycans are required for thrombin-mediated activation of pro-MMP-2. A , Western blot analysis using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-was 2 incubated with 50 n m thrombin in HBMEC cells for the indicated times. Cells
Figure Legend Snippet: Heparan sulfate proteoglycans are required for thrombin-mediated activation of pro-MMP-2. A , Western blot analysis using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-was 2 incubated with 50 n m thrombin in HBMEC cells for the indicated times. Cells

Techniques Used: Activation Assay, Western Blot, Incubation

Binding of heparan sulfate to thrombin elicits a decrease in thrombin-mediated MMP-2 degradation and a concomitant increase in activation. A , shown is the effect of heparan sulfate on the activation and degradation of MMP-2 and fibrinogen cleavage by
Figure Legend Snippet: Binding of heparan sulfate to thrombin elicits a decrease in thrombin-mediated MMP-2 degradation and a concomitant increase in activation. A , shown is the effect of heparan sulfate on the activation and degradation of MMP-2 and fibrinogen cleavage by

Techniques Used: Binding Assay, Activation Assay

Simultaneous binding of MMP-2 to exosite 1 and 2 is essential for its degradation by thrombin. A , Western blot ( IB ) analysis using an anti-myc antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate (40
Figure Legend Snippet: Simultaneous binding of MMP-2 to exosite 1 and 2 is essential for its degradation by thrombin. A , Western blot ( IB ) analysis using an anti-myc antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate (40

Techniques Used: Binding Assay, Western Blot, Incubation

Expression of syndecan-1 increases thrombin-mediated activation of pro-MMP-2 in K562 cells. A , flow cytometric analysis shows cell surface expression of heparan sulfate in K562 cells stably transfected with syndecan-1 is shown. A sample lacking primary
Figure Legend Snippet: Expression of syndecan-1 increases thrombin-mediated activation of pro-MMP-2 in K562 cells. A , flow cytometric analysis shows cell surface expression of heparan sulfate in K562 cells stably transfected with syndecan-1 is shown. A sample lacking primary

Techniques Used: Expressing, Activation Assay, Flow Cytometry, Stable Transfection, Transfection

Heparan sulfate increases thrombin-mediated activation of pro-MMP-2 under cell-free conditions. A , Western blotting using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate under cell-free
Figure Legend Snippet: Heparan sulfate increases thrombin-mediated activation of pro-MMP-2 under cell-free conditions. A , Western blotting using an anti-MMP-2 antibody of 20 μg/ml pro-MMP-2 incubated with 50 n m thrombin in the presence of heparan sulfate under cell-free

Techniques Used: Activation Assay, Western Blot, Incubation

23) Product Images from "Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α"

Article Title: Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α

Journal: PLoS ONE

doi: 10.1371/journal.pone.0105652

Downregulation of EF1α in various types of host cell infected with O. tsutsugamushi . (A) The results of immunoblot analysis of the total cellular protein isolated from ECV304 cells infected with O. tsutsugamushi at the indicated time points are shown. Protein levels were standardized to GAPDH, and the level of TSA56, a type-specific antigen of O. tsutsugamushi , was monitored to confirm bacterial replication during the infection periods. EF1α levels during the infection were analyzed using anti-EF1α antibody. (B) EF1α levels were analyzed in lysates prepared from different types of cell lines at 2 d after the infection with O. tsutsugamushi and compared with that of uninfected cells. GAPDH and TSA56 were monitored simultaneously as protein loading and bacterial infection controls, respectively. UI, uninfected; I, infected. (C) EF1α levels were analyzed in HeLa cells treated with MG132 (10 µM) for 4 h at 2 d after infection with O. tsutsugamushi . GAPDH was used as protein loading control. (D) EF1α mRNA levels were examined by real-time RT-PCR and normalized to β-actin mRNAs in HeLa cells infected with O. tsutsugamushi . The data are presented as mean+SD of three independent experiments.
Figure Legend Snippet: Downregulation of EF1α in various types of host cell infected with O. tsutsugamushi . (A) The results of immunoblot analysis of the total cellular protein isolated from ECV304 cells infected with O. tsutsugamushi at the indicated time points are shown. Protein levels were standardized to GAPDH, and the level of TSA56, a type-specific antigen of O. tsutsugamushi , was monitored to confirm bacterial replication during the infection periods. EF1α levels during the infection were analyzed using anti-EF1α antibody. (B) EF1α levels were analyzed in lysates prepared from different types of cell lines at 2 d after the infection with O. tsutsugamushi and compared with that of uninfected cells. GAPDH and TSA56 were monitored simultaneously as protein loading and bacterial infection controls, respectively. UI, uninfected; I, infected. (C) EF1α levels were analyzed in HeLa cells treated with MG132 (10 µM) for 4 h at 2 d after infection with O. tsutsugamushi . GAPDH was used as protein loading control. (D) EF1α mRNA levels were examined by real-time RT-PCR and normalized to β-actin mRNAs in HeLa cells infected with O. tsutsugamushi . The data are presented as mean+SD of three independent experiments.

Techniques Used: Infection, Isolation, Quantitative RT-PCR

Identification of cellular proteins that interact with Ank proteins. (A) Glutathione-Sepharose beads containing GST or one of the nine Ank proteins fused with GST were mixed with ECV304 cell lysate. Cellular interacting proteins were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Arrows indicate Cullin1 and EF1α, which were identified by mass spectrometry. (B) Immunoblot analyses were performed using specific antibodies and the cellular protein precipitates obtained from GST pull-down assays. At the bottom of the image, GST and the recombinant Ank proteins used in the pull-down assays are visualized after Coomassie brilliant blue (CBB).
Figure Legend Snippet: Identification of cellular proteins that interact with Ank proteins. (A) Glutathione-Sepharose beads containing GST or one of the nine Ank proteins fused with GST were mixed with ECV304 cell lysate. Cellular interacting proteins were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Arrows indicate Cullin1 and EF1α, which were identified by mass spectrometry. (B) Immunoblot analyses were performed using specific antibodies and the cellular protein precipitates obtained from GST pull-down assays. At the bottom of the image, GST and the recombinant Ank proteins used in the pull-down assays are visualized after Coomassie brilliant blue (CBB).

Techniques Used: SDS Page, Staining, Mass Spectrometry, Recombinant

Ank1U5-mediated ubiquitination and downregulation of EF1α. (A) HeLa cells transfected with Flag-Ank1U5 for 18 h were fixed, permeabilized, and stained with anti-Flag antibody together with anti-Cullin1 or anti-EF1α antibody. Merged images show the colocalization of Ank1U5 (red) with endogenous Cullin1 and EF1α (green) in the nucleus. (B) HeLa cells transfected with either vector DNA or plasmid encoding Flag-Ank1U5 were subjected to immunoblot to monitor EF1α levels 48 h after transfection. Levels of GAPDH indicate equal protein loading. (C) The effect of Ank1U5 on EF1α ubiquitination was examined by in vitro ubiquitination reaction. EF1α was immunoprecipitated from the reaction mixture in the absence or presence of the recombinant GST-Ank1U5 at the indicated amounts and subjected to immunoblotting with anti-ubiquitin antibody.
Figure Legend Snippet: Ank1U5-mediated ubiquitination and downregulation of EF1α. (A) HeLa cells transfected with Flag-Ank1U5 for 18 h were fixed, permeabilized, and stained with anti-Flag antibody together with anti-Cullin1 or anti-EF1α antibody. Merged images show the colocalization of Ank1U5 (red) with endogenous Cullin1 and EF1α (green) in the nucleus. (B) HeLa cells transfected with either vector DNA or plasmid encoding Flag-Ank1U5 were subjected to immunoblot to monitor EF1α levels 48 h after transfection. Levels of GAPDH indicate equal protein loading. (C) The effect of Ank1U5 on EF1α ubiquitination was examined by in vitro ubiquitination reaction. EF1α was immunoprecipitated from the reaction mixture in the absence or presence of the recombinant GST-Ank1U5 at the indicated amounts and subjected to immunoblotting with anti-ubiquitin antibody.

Techniques Used: Transfection, Staining, Plasmid Preparation, In Vitro, Immunoprecipitation, Recombinant

24) Product Images from "Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology"

Article Title: Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201008050

Sarm1 is widely expressed in rodent brain and neurons. (A) Immunoblot of Sarm1 in different mouse organs. GAPDH is used as an internal control. (B) Regional distribution of Sarm1 in mouse brain. Cx, cerebral cortex; Hi, hippocampus; St, striatum; Th, thalamus; Cb, cerebellum; BS, brain stem. α-Tubulin was used as an internal control. (C) Staining patterns of Sarm1 in mouse brain. The top right shows the merged image of the MAP2/Sarm1 double stain in the CA1 region of the hippocampus. The top left and bottom panels depict the Sarm1 patterns in brain regions including layer five of the somatosensory cortex (Cx), the posterior thalamic nuclear group (Th), and the caudate putamen of the striatum (St). 2-mo-old mice were used in A–C. (D) Developmental expression profile of Sarm1. The plotted relative Sarm1 protein expression levels were obtained by normalization to the corresponding α-tubulin protein amounts. The results are the means of three independent experiments. Error bars indicate SEM. (E) Distribution of Sarm1 protein in biochemical subcellular fractions of adult mouse brain. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2, crude synaptosomal fraction; S2, supernatant of P2; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosolic fraction. PSD-95 enriched in the P2 and LP1 fractions was used as a quality control of fraction preparation. Molecular mass standards (kD) are indicated next to the gel blots. (F) Distribution of PSD-95 (red) and Sarm1 (green) in cultured hippocampal neurons at 21 DIV. Representative high-magnification images are shown on the top right. Arrowheads indicate the Sarm1 puncta overlapping with PSD-95; arrows indicate the Sarm1 puncta adjacent to PSD-95 puncta. The percentage of overlapped Sarm1 and PSD-95 is shown on the bottom right. The original images and the overlays shifted for 1 and 1.66 µm were analyzed. Error bars indicate mean values ± SEM. **, P
Figure Legend Snippet: Sarm1 is widely expressed in rodent brain and neurons. (A) Immunoblot of Sarm1 in different mouse organs. GAPDH is used as an internal control. (B) Regional distribution of Sarm1 in mouse brain. Cx, cerebral cortex; Hi, hippocampus; St, striatum; Th, thalamus; Cb, cerebellum; BS, brain stem. α-Tubulin was used as an internal control. (C) Staining patterns of Sarm1 in mouse brain. The top right shows the merged image of the MAP2/Sarm1 double stain in the CA1 region of the hippocampus. The top left and bottom panels depict the Sarm1 patterns in brain regions including layer five of the somatosensory cortex (Cx), the posterior thalamic nuclear group (Th), and the caudate putamen of the striatum (St). 2-mo-old mice were used in A–C. (D) Developmental expression profile of Sarm1. The plotted relative Sarm1 protein expression levels were obtained by normalization to the corresponding α-tubulin protein amounts. The results are the means of three independent experiments. Error bars indicate SEM. (E) Distribution of Sarm1 protein in biochemical subcellular fractions of adult mouse brain. H, total homogenate; P1, nuclei and cell debris; S1, supernatant of P1; P2, crude synaptosomal fraction; S2, supernatant of P2; LP1, lysed synaptosomal membrane; LS1, supernatant of LP1; P3, light membrane fraction; S3, soluble cytosolic fraction. PSD-95 enriched in the P2 and LP1 fractions was used as a quality control of fraction preparation. Molecular mass standards (kD) are indicated next to the gel blots. (F) Distribution of PSD-95 (red) and Sarm1 (green) in cultured hippocampal neurons at 21 DIV. Representative high-magnification images are shown on the top right. Arrowheads indicate the Sarm1 puncta overlapping with PSD-95; arrows indicate the Sarm1 puncta adjacent to PSD-95 puncta. The percentage of overlapped Sarm1 and PSD-95 is shown on the bottom right. The original images and the overlays shifted for 1 and 1.66 µm were analyzed. Error bars indicate mean values ± SEM. **, P

Techniques Used: Staining, Mouse Assay, Expressing, Cell Culture

25) Product Images from "Vimentin-Mediated Steroidogenesis Induced by Phthalate Esters: Involvement of DNA Demethylation and Nuclear Factor κB"

Article Title: Vimentin-Mediated Steroidogenesis Induced by Phthalate Esters: Involvement of DNA Demethylation and Nuclear Factor κB

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146138

Effects of MBP and hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to1000 nM MBP in the absence or presence of 100 U/L hCG or 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p
Figure Legend Snippet: Effects of MBP and hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to1000 nM MBP in the absence or presence of 100 U/L hCG or 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p

Techniques Used: Western Blot

Effects of MBP on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were treated by 1000 nM MBP for 24 h. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p
Figure Legend Snippet: Effects of MBP on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were treated by 1000 nM MBP for 24 h. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p

Techniques Used: Western Blot

Effects of hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to 100 U/L hCG or to 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. *p
Figure Legend Snippet: Effects of hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin. MLTC-1 and Y1 cells were exposed to 100 U/L hCG or to 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. *p

Techniques Used: Western Blot

26) Product Images from "Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival"

Article Title: Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00937-14

Syk expression protects MDA-MB-231 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MDA-MB-231-TR (TR) cells lacking Syk or MDA-MB-231-TRS (TRS) cells either induced (+) or not induced (−) with doxycycline (Tet) to express Syk-EGFP were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (B) Comparison of relative levels of Bcl-x L mRNA to Bcl-x S mRNA. Ratios were normalized to a value of 1.0 for Syk-deficient cells at time zero. Bars represent means ± SEMs from three replicate experiments. *, P
Figure Legend Snippet: Syk expression protects MDA-MB-231 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MDA-MB-231-TR (TR) cells lacking Syk or MDA-MB-231-TRS (TRS) cells either induced (+) or not induced (−) with doxycycline (Tet) to express Syk-EGFP were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (B) Comparison of relative levels of Bcl-x L mRNA to Bcl-x S mRNA. Ratios were normalized to a value of 1.0 for Syk-deficient cells at time zero. Bars represent means ± SEMs from three replicate experiments. *, P

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

Nucleolin is required for Syk-dependent stabilization of Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells were untreated (control [Ctrl]) or infected with one of a set of lentiviruses encoding shRNAs for nucleolin (shNCL). Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. Results from three different populations of infected cells are shown. (B) Tet-responsive MDA-MB-231 cells and two of the three sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL2 and shNCL3) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (C) DG75 B cells were untreated (control) or infected with a lentivirus encoding shRNA directed against nucleolin. Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. (D) DG75 cells either infected (+) or not infected (−) with the lentivirus carrying the nucleolin shRNA were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (E) Tet-responsive MCF7 cells were untreated (control) or infected with a set of lentiviruses encoding shRNAs for nucleolin. Nucleolin levels were measured by Western blotting. Results from two different populations of infected cells are shown. (F) Tet-responsive MCF7 cells and the two sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL1 and shNCL2) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA.
Figure Legend Snippet: Nucleolin is required for Syk-dependent stabilization of Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells were untreated (control [Ctrl]) or infected with one of a set of lentiviruses encoding shRNAs for nucleolin (shNCL). Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. Results from three different populations of infected cells are shown. (B) Tet-responsive MDA-MB-231 cells and two of the three sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL2 and shNCL3) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (C) DG75 B cells were untreated (control) or infected with a lentivirus encoding shRNA directed against nucleolin. Nucleolin levels were measured by Western blotting. The level of GAPDH was measured as a loading control. (D) DG75 cells either infected (+) or not infected (−) with the lentivirus carrying the nucleolin shRNA were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA. (E) Tet-responsive MCF7 cells were untreated (control) or infected with a set of lentiviruses encoding shRNAs for nucleolin. Nucleolin levels were measured by Western blotting. Results from two different populations of infected cells are shown. (F) Tet-responsive MCF7 cells and the two sets of Tet-responsive cells carrying the nucleolin shRNA (shNCL1 and shNCL2) were either uninduced (−) or induced with doxycycline to express Syk-EGFP (+) and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA.

Techniques Used: Multiple Displacement Amplification, Infection, Western Blot, shRNA, Reverse Transcription Polymerase Chain Reaction

Syk interacts with nucleolin. (A) MDA-MB-231 cells expressing rtTA but not Syk or MDA-MB-231 cells with Tet-regulated expression of Syk-EGFP or Syk-EGFP(K396R) (Lenti-X Tet-On) pretreated with doxycycline (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting with anti-Syk antibodies to detect Syk-EGFP or Syk-EGFP(K396R) (bottom). (B) Tet-responsive MDA-MB-231 cells not induced (−) or induced with doxycycline to express Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Syk-EGFP was immunoprecipitated (IP) from cell lysates with GFP-nanotrap beads. Anti-GFP immune complexes were separated by SDS-PAGE and analyzed by Western blotting (WB) with antibodies against NCL, γ-tubulin, or GFP (to detect Syk-EGFP). Whole-cell lysates (WCL) were analyzed by Western blotting with antibodies against phosphotyrosine (pTyr) (bottom). The migration position of the 50-kDa molecular mass marker is indicated. (C) Syk-EGFP was immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP. Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL or γ-tubulin. (D) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) and, to detect Syk-GFP, antibodies against GFP (bottom). (E) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express EGFP (lane GFP), Syk-EGFP (lane Syk), Syk-EGFP(Y342F/Y346F) (lane 2F), Syk-EGFP(Y317F/Y342F/Y346F) (lane 3F), Syk-EGFP(Y317F) (lane 317), Syk-EGFP(Y342F) (lane 342), or Syk-EGFP(Y346F) (lane 346) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) or GFP (bottom).
Figure Legend Snippet: Syk interacts with nucleolin. (A) MDA-MB-231 cells expressing rtTA but not Syk or MDA-MB-231 cells with Tet-regulated expression of Syk-EGFP or Syk-EGFP(K396R) (Lenti-X Tet-On) pretreated with doxycycline (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting with anti-Syk antibodies to detect Syk-EGFP or Syk-EGFP(K396R) (bottom). (B) Tet-responsive MDA-MB-231 cells not induced (−) or induced with doxycycline to express Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Syk-EGFP was immunoprecipitated (IP) from cell lysates with GFP-nanotrap beads. Anti-GFP immune complexes were separated by SDS-PAGE and analyzed by Western blotting (WB) with antibodies against NCL, γ-tubulin, or GFP (to detect Syk-EGFP). Whole-cell lysates (WCL) were analyzed by Western blotting with antibodies against phosphotyrosine (pTyr) (bottom). The migration position of the 50-kDa molecular mass marker is indicated. (C) Syk-EGFP was immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP. Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL or γ-tubulin. (D) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) and, to detect Syk-GFP, antibodies against GFP (bottom). (E) Proteins were immunoprecipitated with GFP-nanotrap beads from lysates of Tet-responsive MDA-MB-231 cells induced to express EGFP (lane GFP), Syk-EGFP (lane Syk), Syk-EGFP(Y342F/Y346F) (lane 2F), Syk-EGFP(Y317F/Y342F/Y346F) (lane 3F), Syk-EGFP(Y317F) (lane 317), Syk-EGFP(Y342F) (lane 342), or Syk-EGFP(Y346F) (lane 346) and treated with (+) or without (−) 5 mM H 2 O 2 . Immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL (top) or GFP (bottom).

Techniques Used: Multiple Displacement Amplification, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, SDS Page, Migration, Marker

Syk expression protects MCF7 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against PARP (top). The cleaved form of PARP is indicated by the arrow. The expression of Syk-EGFP was visualized by Western blotting of cell lysates (bottom). (B) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were exposed to 5 mM H 2 O 2 for 30 min (pulse) and then moved to fresh medium for the indicated total incubation times or treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (C) Comparison of relative levels of Bcl-x L mRNA. Changes in the ratio of Bcl-x L mRNA to Bcl-x S mRNA were normalized to their relative levels of expression in Syk-deficient cells at time zero, which was set equal to a value of 1.0. Bars represent means ± SEMs from three replicate experiments. Significant differences between pairs were determined using an unpaired, two-tailed Student's t test. *, P
Figure Legend Snippet: Syk expression protects MCF7 cells from oxidative stress-induced apoptosis and degradation of Bcl-x L mRNA. (A) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against PARP (top). The cleaved form of PARP is indicated by the arrow. The expression of Syk-EGFP was visualized by Western blotting of cell lysates (bottom). (B) MCF7-BD cells lacking Syk (−) or stably expressing Syk-EGFP (+) were exposed to 5 mM H 2 O 2 for 30 min (pulse) and then moved to fresh medium for the indicated total incubation times or treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were analyzed by RT-PCR to measure the levels of Bcl-x L and Bcl-x S mRNA (top) or by Western blotting to detect expressed Syk-EGFP (bottom). (C) Comparison of relative levels of Bcl-x L mRNA. Changes in the ratio of Bcl-x L mRNA to Bcl-x S mRNA were normalized to their relative levels of expression in Syk-deficient cells at time zero, which was set equal to a value of 1.0. Bars represent means ± SEMs from three replicate experiments. Significant differences between pairs were determined using an unpaired, two-tailed Student's t test. *, P

Techniques Used: Expressing, Stable Transfection, SDS Page, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Nucleolin is required for Syk-dependent protection of cells from stress-induced apoptosis. (A) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (B) DG75 cells or DG75 cells expressing the shRNA targeting either Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (C) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 1 μg/ml doxorubicin (Dox) for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (D) DG75 cells or DG75 cells expressing either the shRNA targeting Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 1 μg/ml doxorubicin for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (E) The degree of PARP cleavage was quantified from Western blots of lysates of MCF7 cells lacking Syk (no Syk), expressing Syk-EGFP (SykGFP), or expressing Syk-EGFP and shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H 2 O 2 (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. **, P
Figure Legend Snippet: Nucleolin is required for Syk-dependent protection of cells from stress-induced apoptosis. (A) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (B) DG75 cells or DG75 cells expressing the shRNA targeting either Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 5 mM H 2 O 2 for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (C) Tet-responsive MCF7 cells or Tet-responsive cells carrying the nucleolin shRNA were either uninduced or induced with doxycycline to express Syk-EGFP and then treated with 1 μg/ml doxorubicin (Dox) for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (D) DG75 cells or DG75 cells expressing either the shRNA targeting Syk (Syk-shRNA) or nucleolin (NCL-shRNA) were treated with 1 μg/ml doxorubicin for the indicated times. Cell lysates were separated by SDS-PAGE and probed with antibodies against PARP (top), nucleolin (middle), or Syk (bottom). The cleaved form of PARP is indicated by the arrow. (E) The degree of PARP cleavage was quantified from Western blots of lysates of MCF7 cells lacking Syk (no Syk), expressing Syk-EGFP (SykGFP), or expressing Syk-EGFP and shRNA for nucleolin (shNCL) and treated for 24 h with 5 mM H 2 O 2 (left) or 1 μg/ml doxorubicin (right). The data represent means ± SEMs from three replicate experiments. **, P

Techniques Used: shRNA, SDS Page, Expressing, Western Blot

Syk phosphorylates nucleolin and promotes its binding to Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells pretreated without or with doxycycline to induce Syk-EGFP were treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (WCL; bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (B) DG75 B cells were pretreated with 50 μM piceatannol (PIC; +) or dimethyl sulfoxide carrier alone (−) and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (C) DG75 cells were pretreated with the indicated concentrations of R406 and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (D) MDA-MB-231-TR (TR) or MDA-MB-231-TRS cells induced to express Syk-EGFP (TRS) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (E) DG75 B cells (−) or DG75 cells stably expressing shRNA targeted against Syk (+) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (F) Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) was immunoprecipitated from the corresponding doxycycline-induced lines of MDA-MB-231 cells using GFP-nanotrap beads. The resulting immune complexes were incubated with buffer containing (+) or lacking (−) ATP. The immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (G) Nucleolin was immunoprecipitated from Tet-responsive MDA-MB-231 cells either uninduced (−) or induced (+) with doxycycline to express Syk-EGFP and either treated with 5 mM H 2 O 2 for 3 h or not treated. Immune complexes were examined for the presence of Bcl-x L mRNA by RT-PCR (top) and nucleolin by Western blotting (middle). The expression of Syk-EGFP was determined by Western blotting of whole-cell lysates with antibodies against Syk (bottom). (H) The relative amount of Bcl-x L mRNA associated with nucleolin, analyzed as described in the legend to panel G, was quantified. The data represent means ± SEMs from three replicate experiments. The level of mRNA bound to nucleolin in Syk-EGFP-expressing cells not treated with H 2 O 2 was set equal to a value of 1.0.
Figure Legend Snippet: Syk phosphorylates nucleolin and promotes its binding to Bcl-x L mRNA. (A) Tet-responsive MDA-MB-231 cells pretreated without or with doxycycline to induce Syk-EGFP were treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (WCL; bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (B) DG75 B cells were pretreated with 50 μM piceatannol (PIC; +) or dimethyl sulfoxide carrier alone (−) and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (C) DG75 cells were pretreated with the indicated concentrations of R406 and then treated without or with 5 mM H 2 O 2 for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (D) MDA-MB-231-TR (TR) or MDA-MB-231-TRS cells induced to express Syk-EGFP (TRS) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (E) DG75 B cells (−) or DG75 cells stably expressing shRNA targeted against Syk (+) were treated without or with 5 mM H 2 O 2 for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (F) Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) was immunoprecipitated from the corresponding doxycycline-induced lines of MDA-MB-231 cells using GFP-nanotrap beads. The resulting immune complexes were incubated with buffer containing (+) or lacking (−) ATP. The immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (G) Nucleolin was immunoprecipitated from Tet-responsive MDA-MB-231 cells either uninduced (−) or induced (+) with doxycycline to express Syk-EGFP and either treated with 5 mM H 2 O 2 for 3 h or not treated. Immune complexes were examined for the presence of Bcl-x L mRNA by RT-PCR (top) and nucleolin by Western blotting (middle). The expression of Syk-EGFP was determined by Western blotting of whole-cell lysates with antibodies against Syk (bottom). (H) The relative amount of Bcl-x L mRNA associated with nucleolin, analyzed as described in the legend to panel G, was quantified. The data represent means ± SEMs from three replicate experiments. The level of mRNA bound to nucleolin in Syk-EGFP-expressing cells not treated with H 2 O 2 was set equal to a value of 1.0.

Techniques Used: Binding Assay, Multiple Displacement Amplification, Immunoprecipitation, SDS Page, Western Blot, Stable Transfection, Expressing, shRNA, Incubation, Reverse Transcription Polymerase Chain Reaction

27) Product Images from "Hairy and Enhancer of Split-related with YRPW Motif (HEY)2 Regulates Bone Remodeling in Mice"

Article Title: Hairy and Enhancer of Split-related with YRPW Motif (HEY)2 Regulates Bone Remodeling in Mice

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.489435

HEY2 overexpression suppresses osteoblast function in vitro . A and B , osteoblast-enriched cells were harvested from the calvariae of male ( A ) or female ( B ) Col3.6-Hey2 transgenic mice ( Hey2 , black bars ) or littermate wild type controls of the same sex
Figure Legend Snippet: HEY2 overexpression suppresses osteoblast function in vitro . A and B , osteoblast-enriched cells were harvested from the calvariae of male ( A ) or female ( B ) Col3.6-Hey2 transgenic mice ( Hey2 , black bars ) or littermate wild type controls of the same sex

Techniques Used: Over Expression, In Vitro, Transgenic Assay, Mouse Assay

Hey2 inactivation inhibits osteoblast function in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Oc-Cre +/− ; Hey2 Δ/Δ mice ( Hey2 null , black bars ), or littermate Hey2 loxP/loxP controls of the same
Figure Legend Snippet: Hey2 inactivation inhibits osteoblast function in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Oc-Cre +/− ; Hey2 Δ/Δ mice ( Hey2 null , black bars ), or littermate Hey2 loxP/loxP controls of the same

Techniques Used: In Vitro, Mouse Assay

HEY2 overexpression in osteoblasts from male mice induces resorption by co-cultured splenocytes and IL6 expression in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Col3.6-Hey2 transgenics ( Hey2 , black bars ), or littermate
Figure Legend Snippet: HEY2 overexpression in osteoblasts from male mice induces resorption by co-cultured splenocytes and IL6 expression in vitro . Osteoblast-enriched cells were harvested from calvariae of male or female Col3.6-Hey2 transgenics ( Hey2 , black bars ), or littermate

Techniques Used: Over Expression, Mouse Assay, Cell Culture, Expressing, In Vitro

28) Product Images from "Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamateric synapses"

Article Title: Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamateric synapses

Journal: Journal of neurochemistry

doi: 10.1111/jnc.12436

Dependence of the ACM from nicotine-treated astrocyte (A/Nic) effect on astrocyte α7-nAChRs, longevity of the induced increase in GluA1 puncta, and the continuing responsiveness of cultures. (a) A/Nic obtained from astrocytes treated with methyllycaconitine
Figure Legend Snippet: Dependence of the ACM from nicotine-treated astrocyte (A/Nic) effect on astrocyte α7-nAChRs, longevity of the induced increase in GluA1 puncta, and the continuing responsiveness of cultures. (a) A/Nic obtained from astrocytes treated with methyllycaconitine

Techniques Used:

ACM from nicotine-treated astrocyte (A/Nic) increases the number of GluA1 and GluA2 puncta on the neuron surface without changing the number of VGluT, post-synaptic density protein 95 (PSD-95), or NR1 puncta. One-week-old hippocampal cultures were incubated
Figure Legend Snippet: ACM from nicotine-treated astrocyte (A/Nic) increases the number of GluA1 and GluA2 puncta on the neuron surface without changing the number of VGluT, post-synaptic density protein 95 (PSD-95), or NR1 puncta. One-week-old hippocampal cultures were incubated

Techniques Used: Incubation

29) Product Images from "Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells"

Article Title: Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells

Journal: World Journal of Gastroenterology : WJG

doi: 10.3748/wjg.v19.i25.3969

The effect of BN52021 on platelet-activating factor receptor signaling molecules at the mRNA level under lipopolysaccharide-induced inflammation. The mRNA level of adenylate cyclase (AC) (A), G protein-coupled receptor kinases (GRK) (B), phospholipase A 2 (PLA 2 ) (C), phospholipase Cβ (PLCβ) (D), p38-mitogen-activated protein kinase (p38 MAPK) (E) and protein tyrosine kinase (PTK) (F) was up-regulated after lipopolysaccharide (LPS) stimulation. The up-regulation of AC, GRK, p38 MAPK, PLCβ and PLA 2 mRNA was significantly suppressed by BN52021 except for that of PTK. a P
Figure Legend Snippet: The effect of BN52021 on platelet-activating factor receptor signaling molecules at the mRNA level under lipopolysaccharide-induced inflammation. The mRNA level of adenylate cyclase (AC) (A), G protein-coupled receptor kinases (GRK) (B), phospholipase A 2 (PLA 2 ) (C), phospholipase Cβ (PLCβ) (D), p38-mitogen-activated protein kinase (p38 MAPK) (E) and protein tyrosine kinase (PTK) (F) was up-regulated after lipopolysaccharide (LPS) stimulation. The up-regulation of AC, GRK, p38 MAPK, PLCβ and PLA 2 mRNA was significantly suppressed by BN52021 except for that of PTK. a P

Techniques Used: Proximity Ligation Assay

The effect of BN52021 on platelet-activating factor receptor signaling molecules at the protein level under lipopolysaccharide-induced inflammation. The protein level of p-adenylate cyclase (p-AC) (A), G protein-coupled receptor kinases (GRK) (B), p-phospholipase A 2 (p-PLA 2 ) (C), phospholipase Cβ (PLCβ) (D) and p-p38-mitogen-activated protein kinase (p-p38 MAPK) (E) was up-regulated after lipopolysaccharide (LPS) stimulation vs the blank control ( a P
Figure Legend Snippet: The effect of BN52021 on platelet-activating factor receptor signaling molecules at the protein level under lipopolysaccharide-induced inflammation. The protein level of p-adenylate cyclase (p-AC) (A), G protein-coupled receptor kinases (GRK) (B), p-phospholipase A 2 (p-PLA 2 ) (C), phospholipase Cβ (PLCβ) (D) and p-p38-mitogen-activated protein kinase (p-p38 MAPK) (E) was up-regulated after lipopolysaccharide (LPS) stimulation vs the blank control ( a P

Techniques Used: Proximity Ligation Assay

The dose effect of BN52021 on lipopolysaccharide-induced inflammation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and Hoechst 33342/propidium iodide staining. MS1 cell activity at A 490 nm was significantly decreased 24 h after administration of 10 μg/mL lipopolysaccharide (LPS) vs the control group ( a P
Figure Legend Snippet: The dose effect of BN52021 on lipopolysaccharide-induced inflammation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and Hoechst 33342/propidium iodide staining. MS1 cell activity at A 490 nm was significantly decreased 24 h after administration of 10 μg/mL lipopolysaccharide (LPS) vs the control group ( a P

Techniques Used: Staining, Activity Assay

30) Product Images from "Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc"

Article Title: Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E07-07-0690

Knockdown of nebulin in primary cultures of chick skeletal myotubes results in a loss of CapZ at the Z-disc. (A) Myotubes were triple stained with antibodies to N-terminal nebulin, CapZ, and α-actinin 3 d after siRNA treatment. Treatment with nebulin-specific siRNA resulted in a dramatic decrease in nebulin staining and a reduction in the amount of CapZ at the Z-disc, with α-actinin only partially perturbed. (B) Knockdown of nebulin also resulted in phalloidin staining along the entire thin filament, not just at the Z-disc (arrowheads) and pointed ends (arrows) as observed in control cells. Myotubes were costained with an anti-C-terminal nebulin antibody. Bar, 10 μm. (C) RT-PCR consistently showed a ≥70% reduction in nebulin transcript levels in chick skeletal myotubes 1 d after treatment with nebulin-specific (KD) versus control (C) siRNA, whereas GAPDH transcript levels were similar in both samples. (D) Western blot analysis revealed a ≥90% decrease in nebulin protein levels (arrow) in chick skeletal myotubes two days after treatment with nebulin-specific (KD) versus control (C) siRNA by using an anti-N-terminal nebulin antibody.
Figure Legend Snippet: Knockdown of nebulin in primary cultures of chick skeletal myotubes results in a loss of CapZ at the Z-disc. (A) Myotubes were triple stained with antibodies to N-terminal nebulin, CapZ, and α-actinin 3 d after siRNA treatment. Treatment with nebulin-specific siRNA resulted in a dramatic decrease in nebulin staining and a reduction in the amount of CapZ at the Z-disc, with α-actinin only partially perturbed. (B) Knockdown of nebulin also resulted in phalloidin staining along the entire thin filament, not just at the Z-disc (arrowheads) and pointed ends (arrows) as observed in control cells. Myotubes were costained with an anti-C-terminal nebulin antibody. Bar, 10 μm. (C) RT-PCR consistently showed a ≥70% reduction in nebulin transcript levels in chick skeletal myotubes 1 d after treatment with nebulin-specific (KD) versus control (C) siRNA, whereas GAPDH transcript levels were similar in both samples. (D) Western blot analysis revealed a ≥90% decrease in nebulin protein levels (arrow) in chick skeletal myotubes two days after treatment with nebulin-specific (KD) versus control (C) siRNA by using an anti-N-terminal nebulin antibody.

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

31) Product Images from "Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket"

Article Title: Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket

Journal: eLife

doi: 10.7554/eLife.18492

Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004
Figure Legend Snippet: Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004

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

32) Product Images from "Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton"

Article Title: Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126214

Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P
Figure Legend Snippet: Distinct Tpm isoforms differentially impact on the elastic modulus of the cell. Tpm-overexpressing clones were generated by the stable transfection of Tpm containing vectors. (A) 10 μg of total cellular protein isolated from the Tpm- clones was analysed by SDS-PAGE followed by western blotting. Shown are representative blots probed with the Tm311 (detecting Tpm2.1, Tpm1.10, Tpm1.7), α/9b (Tpm1.11), α/9c (Tpm1.10, Tpm1.12), δ/9d (Tpm4.2), γ/9d (Tpm3.1), and GAPDH antibodies. (B) The elastic (Young) modulus for each Tpm-overexpressing clone was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. 12–25 cells for each clone was measured from n = 3 independent experiments. * P

Techniques Used: Clone Assay, Generated, Stable Transfection, Isolation, SDS Page, Western Blot, Whisker Assay

33) 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

34) Product Images from "SLC25A22 Promotes Proliferation and Metastasis of Osteosarcoma Cells via the PTEN Signaling Pathway"

Article Title: SLC25A22 Promotes Proliferation and Metastasis of Osteosarcoma Cells via the PTEN Signaling Pathway

Journal: Technology in Cancer Research & Treatment

doi: 10.1177/1533033818811143

SLC25A22 accelerates cell cycle progression and inhibits cell apoptosis of osteosarcoma cells. A, SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells were subjected to cell cycle detection by flow cytometry. The results were analyzed with Modifit software and the proportions for each period were calculated. B, SLC25A22, cyclin B1, cdc25c, and cyclin D1 protein levels were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells. C, Forty-eight hours after U2OS and Saos-2 cells were transfected with shRNA (SLC25A22 KD or control), cells were stained with FITC-Annexin V and PE-PI, and apoptotic cells were detected by flow cytometry and subsequently analyzed with Flowjo software. D, Cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bad were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells.
Figure Legend Snippet: SLC25A22 accelerates cell cycle progression and inhibits cell apoptosis of osteosarcoma cells. A, SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells were subjected to cell cycle detection by flow cytometry. The results were analyzed with Modifit software and the proportions for each period were calculated. B, SLC25A22, cyclin B1, cdc25c, and cyclin D1 protein levels were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells. C, Forty-eight hours after U2OS and Saos-2 cells were transfected with shRNA (SLC25A22 KD or control), cells were stained with FITC-Annexin V and PE-PI, and apoptotic cells were detected by flow cytometry and subsequently analyzed with Flowjo software. D, Cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bad were detected by Western blot in SLC25A22 KD U2OS, Saos-2 cells and overexpressing HOS cells.

Techniques Used: Flow Cytometry, Cytometry, Software, Western Blot, Transfection, shRNA, Staining

35) Product Images from "Genetic Ablation of Tau Mitigates Cognitive Impairment Induced by Type 1 Diabetes"

Article Title: Genetic Ablation of Tau Mitigates Cognitive Impairment Induced by Type 1 Diabetes

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2013.11.021

Streptozotocin treatment alters the IR/PI3K/AKT pathway in Ntg and tauKO mice. A: Immunoblot analyses of pIR, IR, pPI3k(p85), PI3k, pAKT(Ser473), AKT, GSK3β (Ser9), and GSK3β of protein extracts from whole-brain homogenates of Ntg, Ntg-STZ, tauKO, and tauKO-STZ mice at 5 months of age are shown on alternating lanes. B: Quantification normalized to GAPDH and expressed as percentage of control. Pairwise comparisons: ∗ P
Figure Legend Snippet: Streptozotocin treatment alters the IR/PI3K/AKT pathway in Ntg and tauKO mice. A: Immunoblot analyses of pIR, IR, pPI3k(p85), PI3k, pAKT(Ser473), AKT, GSK3β (Ser9), and GSK3β of protein extracts from whole-brain homogenates of Ntg, Ntg-STZ, tauKO, and tauKO-STZ mice at 5 months of age are shown on alternating lanes. B: Quantification normalized to GAPDH and expressed as percentage of control. Pairwise comparisons: ∗ P

Techniques Used: Mouse Assay

36) Product Images from "Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells"

Article Title: Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

Journal: Molecular Pharmacology

doi: 10.1124/mol.110.064840

Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p
Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p

Techniques Used: Transfection, Activation Assay

Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p
Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p

Techniques Used: Transfection

37) Product Images from "Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells"

Article Title: Bradykinin B2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

Journal: Molecular Pharmacology

doi: 10.1124/mol.110.064840

BK induces complex formation between EGFR and α5β1 integrin. Lysates from mIMCD-3 cells treated with vehicle, 100 nM BK, or 1 ng/ml EGF were immunoprecipitated with anti-α5β1 integrin antibody as described under Materials and Methods . Immunoblotting was performed with antibodies against EGFR (A) and BK B 2 receptor (B). The blots shown are representative of four experiments. A, coimmunoprecipitation experiments show that α5β1 integrin and EGFR coimmunoprecipitate and that their association can be increased by stimulation of mIMCD-3 cells with 100 nM BK but not with EGF. Inset, representative Western blot with antibody against EGFR showing immunoreactive band at 175 kDa. Blot was stripped and re-probed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. B, BK B 2 receptor coimmunoprecipitates with α5β1 integrin. Inset, representative Western blot with antibody against BK B 2 receptor showing immunoreactive duplet at 42/40 kDa. Blot was stripped and reprobed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. IP, immunoprecipitation; IB, immunoblot.
Figure Legend Snippet: BK induces complex formation between EGFR and α5β1 integrin. Lysates from mIMCD-3 cells treated with vehicle, 100 nM BK, or 1 ng/ml EGF were immunoprecipitated with anti-α5β1 integrin antibody as described under Materials and Methods . Immunoblotting was performed with antibodies against EGFR (A) and BK B 2 receptor (B). The blots shown are representative of four experiments. A, coimmunoprecipitation experiments show that α5β1 integrin and EGFR coimmunoprecipitate and that their association can be increased by stimulation of mIMCD-3 cells with 100 nM BK but not with EGF. Inset, representative Western blot with antibody against EGFR showing immunoreactive band at 175 kDa. Blot was stripped and re-probed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. B, BK B 2 receptor coimmunoprecipitates with α5β1 integrin. Inset, representative Western blot with antibody against BK B 2 receptor showing immunoreactive duplet at 42/40 kDa. Blot was stripped and reprobed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. IP, immunoprecipitation; IB, immunoblot.

Techniques Used: Immunoprecipitation, Western Blot

Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p
Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods . Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p

Techniques Used: Transfection, Activation Assay

Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p
Figure Legend Snippet: Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods . Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods . Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p

Techniques Used: Transfection

38) Product Images from "Arl13b and the exocyst interact synergistically in ciliogenesis"

Article Title: Arl13b and the exocyst interact synergistically in ciliogenesis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E15-02-0061

Arl13b interacts with the exocyst in ciliated cells. (A) Endogenous Arl13b was immunoprecipitated from cell lysates of ciliated IMCD3 cells after preincubation with either no nucleotide (left lane), GTPγS (second lane), or GDP (third lane). Nonspecific rabbit IgG was used as a negative control (fourth lane). Input is shown in the far right lane. Immunoprecipitates were resolved by SDS–PAGE and immunoblotted with Sec8 antibody. (B) Cell lysates of ciliated IMCD3 cells were immunoprecipitated with Arl13b after preincubation with GTPγS or GDP. Nonspecific rabbit IgG was used as a negative control. Immunoprecipitates were resolved by SDS–PAGE and immunoblotted with Exo70 or Sec5 antibodies. (C) Total cell lysates of ciliated NIH-3T3 cells were immunoprecipitated and analyzed as described in A. (D) Cell lysates of ciliated IMCD3 cells were immunoprecipitated with Sec8 antibody after incubation with GTPγS. An irrelevant mouse IgG1 (IgG) was used as a negative control. Immunoprecipitates were analyzed by SDS–PAGE and immunoblotted with Arl13b antibody. Results are representative of three independent experiments.
Figure Legend Snippet: Arl13b interacts with the exocyst in ciliated cells. (A) Endogenous Arl13b was immunoprecipitated from cell lysates of ciliated IMCD3 cells after preincubation with either no nucleotide (left lane), GTPγS (second lane), or GDP (third lane). Nonspecific rabbit IgG was used as a negative control (fourth lane). Input is shown in the far right lane. Immunoprecipitates were resolved by SDS–PAGE and immunoblotted with Sec8 antibody. (B) Cell lysates of ciliated IMCD3 cells were immunoprecipitated with Arl13b after preincubation with GTPγS or GDP. Nonspecific rabbit IgG was used as a negative control. Immunoprecipitates were resolved by SDS–PAGE and immunoblotted with Exo70 or Sec5 antibodies. (C) Total cell lysates of ciliated NIH-3T3 cells were immunoprecipitated and analyzed as described in A. (D) Cell lysates of ciliated IMCD3 cells were immunoprecipitated with Sec8 antibody after incubation with GTPγS. An irrelevant mouse IgG1 (IgG) was used as a negative control. Immunoprecipitates were analyzed by SDS–PAGE and immunoblotted with Arl13b antibody. Results are representative of three independent experiments.

Techniques Used: Immunoprecipitation, Negative Control, SDS Page, Incubation

Arl13b interacts directly with Sec5 and Sec8 exocyst subunits. Purified Myc-tagged, in vitro–translated Sec5, Sec8, or Exo70 was mixed with anti–GST-coupled agarose beads previously incubated with purified Arl13b-GST or GST. A negative control (NC) to detect nonspecific binding of exocyst subunits to the agarose beads was added, in which anti–GST-coupled beads were directly incubated with purified Sec8-Myc protein. Eluted products were analyzed by SDS–PAGE, followed by immunoblotting for Myc tag or silver staining. The latter shows that identical amounts of purified Arl13b-GST were incubated in the different conditions. The membrane was previously probed with GST antibody to detect Arl13b-GST (arrowhead). Five percent of the purified proteins used in the assay was run in the input. Results are representative of at least two independent experiments.
Figure Legend Snippet: Arl13b interacts directly with Sec5 and Sec8 exocyst subunits. Purified Myc-tagged, in vitro–translated Sec5, Sec8, or Exo70 was mixed with anti–GST-coupled agarose beads previously incubated with purified Arl13b-GST or GST. A negative control (NC) to detect nonspecific binding of exocyst subunits to the agarose beads was added, in which anti–GST-coupled beads were directly incubated with purified Sec8-Myc protein. Eluted products were analyzed by SDS–PAGE, followed by immunoblotting for Myc tag or silver staining. The latter shows that identical amounts of purified Arl13b-GST were incubated in the different conditions. The membrane was previously probed with GST antibody to detect Arl13b-GST (arrowhead). Five percent of the purified proteins used in the assay was run in the input. Results are representative of at least two independent experiments.

Techniques Used: Purification, In Vitro, Incubation, Negative Control, Binding Assay, SDS Page, Silver Staining

Arl13b interacts with the exocyst through the Sec5 and Sec8 subunits. (A) Sec8-Myc and Arl13b-FLAG were in vitro–translated using TNT T7 coupled reticulocyte lysate system. Five percent of the TNT reaction was resolved by SDS–PAGE and analyzed by immunoblotting with Sec8, Myc, Arl13b, or FLAG antibodies. As a control, a TNT reaction without DNA as template was performed and subsequently used in the immunoprecipitations to distinguish the in vitro–translated from the endogenous protein present in the reticulocyte lysate. (B) Immunoprecipitation with FLAG antibody was performed using the in vitro–translated proteins in the presence of GTPγS. Immunoprecipitates (lane 1) were analyzed by immunoblot with Myc, Sec8, or FLAG antibodies. As a negative control, immunoprecipitations were performed with an irrelevant mouse IgG1 (IgG; lane 2). In addition, as a control, immunoprecipitation with FLAG antibody was performed using as input a mixture of a TNT reaction made without DNA and the in vitro–translated Arl13b-FLAG in the presence of GTPγS (lane 3). Asterisks indicate in vitro–translated Sec8-Myc, and the pound signs indicate endogenous Sec8. (C) Left, Myc-tagged Sec3, Sec5, Sec8, Sec15, and Exo70 exocyst subunits were in vitro translated using the TNT system. Five percent of the TNT reaction was resolved by SDS–PAGE and analyzed by immunoblotting with Myc antibody. Right, endogenous levels of Sec5, Sec8, and Exo70 present on 5% of the TNT reaction were analyzed by immunoblotting with specific antibodies for each subunit. (D) Immunoprecipitation with FLAG antibody was performed using the in vitro–translated proteins described in C, in the presence of GTPγS. Immunoprecipitates were analyzed by immunoblot with Myc antibody or silver staining. Negative controls (lanes 2, 5. and 8) were performed as in B. Results are representative of three independent experiments.
Figure Legend Snippet: Arl13b interacts with the exocyst through the Sec5 and Sec8 subunits. (A) Sec8-Myc and Arl13b-FLAG were in vitro–translated using TNT T7 coupled reticulocyte lysate system. Five percent of the TNT reaction was resolved by SDS–PAGE and analyzed by immunoblotting with Sec8, Myc, Arl13b, or FLAG antibodies. As a control, a TNT reaction without DNA as template was performed and subsequently used in the immunoprecipitations to distinguish the in vitro–translated from the endogenous protein present in the reticulocyte lysate. (B) Immunoprecipitation with FLAG antibody was performed using the in vitro–translated proteins in the presence of GTPγS. Immunoprecipitates (lane 1) were analyzed by immunoblot with Myc, Sec8, or FLAG antibodies. As a negative control, immunoprecipitations were performed with an irrelevant mouse IgG1 (IgG; lane 2). In addition, as a control, immunoprecipitation with FLAG antibody was performed using as input a mixture of a TNT reaction made without DNA and the in vitro–translated Arl13b-FLAG in the presence of GTPγS (lane 3). Asterisks indicate in vitro–translated Sec8-Myc, and the pound signs indicate endogenous Sec8. (C) Left, Myc-tagged Sec3, Sec5, Sec8, Sec15, and Exo70 exocyst subunits were in vitro translated using the TNT system. Five percent of the TNT reaction was resolved by SDS–PAGE and analyzed by immunoblotting with Myc antibody. Right, endogenous levels of Sec5, Sec8, and Exo70 present on 5% of the TNT reaction were analyzed by immunoblotting with specific antibodies for each subunit. (D) Immunoprecipitation with FLAG antibody was performed using the in vitro–translated proteins described in C, in the presence of GTPγS. Immunoprecipitates were analyzed by immunoblot with Myc antibody or silver staining. Negative controls (lanes 2, 5. and 8) were performed as in B. Results are representative of three independent experiments.

Techniques Used: In Vitro, SDS Page, Immunoprecipitation, Negative Control, Silver Staining

39) Product Images from "PHGDH as a key enzyme for serine biosynthesis in HIF2α-targeting therapy for renal cell carcinoma"

Article Title: PHGDH as a key enzyme for serine biosynthesis in HIF2α-targeting therapy for renal cell carcinoma

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-17-1589

PHGDH over expression in 786-o and A498 parental cells A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in 786-o and A498 cells. B, Representative images of parental and PHGDH overexpressed 786-o or A498 cells. C, Immunofluorescence analysis; PHGDH overexpressed 786-o or A498 cells showed morphological changes from a spindle to a round cell shape. The graph showed the ratio between spindle and round cells in PHGDH overexpressed 786-o or A498 cells. D, Representative image of colony formation in parental and PHGDH overexpressed 786-o or A498 cells. The graph showed the ratio of number of colonies between parental and PHGDH overexpressed cells (* P
Figure Legend Snippet: PHGDH over expression in 786-o and A498 parental cells A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in 786-o and A498 cells. B, Representative images of parental and PHGDH overexpressed 786-o or A498 cells. C, Immunofluorescence analysis; PHGDH overexpressed 786-o or A498 cells showed morphological changes from a spindle to a round cell shape. The graph showed the ratio between spindle and round cells in PHGDH overexpressed 786-o or A498 cells. D, Representative image of colony formation in parental and PHGDH overexpressed 786-o or A498 cells. The graph showed the ratio of number of colonies between parental and PHGDH overexpressed cells (* P

Techniques Used: Over Expression, Expressing, Immunofluorescence

Establishment of HIF2α knock out SU-R-786-o cells A, Immunoblotting analysis showed that HIF2α was significantly depleted in sunitinib resistant 786-o cells. B, RSEM values of HIF targets genes from RNA-seq expression data between 786-o empty and HIF2α-KO-SU-R-786-o cells. C, Representative images of HIF2α-KO-SU-R-786-o cells. D, Cell proliferation assay between control and HIF2α-KO-SU-R-786-o (* P
Figure Legend Snippet: Establishment of HIF2α knock out SU-R-786-o cells A, Immunoblotting analysis showed that HIF2α was significantly depleted in sunitinib resistant 786-o cells. B, RSEM values of HIF targets genes from RNA-seq expression data between 786-o empty and HIF2α-KO-SU-R-786-o cells. C, Representative images of HIF2α-KO-SU-R-786-o cells. D, Cell proliferation assay between control and HIF2α-KO-SU-R-786-o (* P

Techniques Used: Knock-Out, RNA Sequencing Assay, Expressing, Proliferation Assay

PHGDH inhibition by si-RNA and inhibitor A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in HIF2α-KO-SU-R-786-o cells. B, Cell proliferation assay by PHGDH si-RNA. C, Cell proliferation assay by PHGDH inhibitor (CBR-5884). (* P
Figure Legend Snippet: PHGDH inhibition by si-RNA and inhibitor A, Immunoblotting analysis showed that PHGDH expression was dramatically elevated in HIF2α-KO-SU-R-786-o cells. B, Cell proliferation assay by PHGDH si-RNA. C, Cell proliferation assay by PHGDH inhibitor (CBR-5884). (* P

Techniques Used: Inhibition, Expressing, Proliferation Assay

40) Product Images from "Effects of Phonation Time and Magnitude Dose on Vocal Fold Epithelial Genes, Barrier Integrity, and Function"

Article Title: Effects of Phonation Time and Magnitude Dose on Vocal Fold Epithelial Genes, Barrier Integrity, and Function

Journal: The Laryngoscope

doi: 10.1002/lary.24827

Gene transcript level changes in the inflammatory mediators Interleukin-1β (IL-1β) and Cyclooxygenase-2 (COX-2), the multifunctional peptide Transforming Growth Factorβ1 (TGFβ1), and the multifunctional extracellular matrix
Figure Legend Snippet: Gene transcript level changes in the inflammatory mediators Interleukin-1β (IL-1β) and Cyclooxygenase-2 (COX-2), the multifunctional peptide Transforming Growth Factorβ1 (TGFβ1), and the multifunctional extracellular matrix

Techniques Used:

Related Articles

Multiplex Assay:

Article Title: An Azabisphosphonate-Capped Poly(phosphorhydrazone) Dendrimer for the Treatment of Endotoxin-Induced Uveitis
Article Snippet: .. Pro-inflammatory T helper cytokines TNFα, IL-1β, IL-2, IL-6, IL-17 and IFNγ as well as anti-inflammatory cytokines IL-4 and IL-10 quantities were determined by Multiplex analysis (Milliplex Map Kit; Millipore, Saint-Quentin-en-Yvelines, France). ..

Incubation:

Article Title: Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice
Article Snippet: .. Finally, for β -actin, the membranes were incubated with anti-β -actin polyclonal antibody (1 : 10000 dilution), obtained from Sigma-Aldrich. .. The blots were washed and incubated with peroxidase-conjugate anti-rabbit, anti-mouse, or anti-goat immunoglobulin G (1 : 12000 dilution; Cayman).

Article Title: A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns *
Article Snippet: .. TAP-tagged proteins were detected by immunoblotting by incubation overnight with an α-calmodulin antibody (Millipore) and for 2 h with α-rabbit IgG HRP-linked antibody (Cell Signaling). .. Gel filtration molecular weight markers (Sigma) were used for determination of the molecular weight range of the fractions.

other:

Article Title: Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization
Article Snippet: SP600125, U0126 and SB202190 were from Calbiochem (La Jolla, CA).

Expressing:

Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
Article Snippet: .. Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

Modification:

Article Title: Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells
Article Snippet: .. Immunoblotting To analyze protein expression, BeWo cells and primary vCTB were rinsed with PBS and lysed with ice-cold modified radioimmunoprecipitation (RIPA) buffer (50 mmol/l Tris-HCl pH 7.4, 1% NP-40, 0,25% Na-deoxycholate, 150 mmol/l NaCl and 1 mmol/l EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich). .. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Waltham, MA).

Recombinant:

Article Title: CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells
Article Snippet: .. Recombinant human CXCL12, U0126, LY294002, and AG1478 were obtained from Sigma-Aldrich. .. PE-conjugated secondary antibody was purchased from R & D systems (Minneapolis, MN).

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    Millipore mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody
    Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to <t>glyceraldehyde-3-phosphate</t> dehydrogenase <t>(GAPDH)</t> at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.
    Mouse Monoclonal Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gapdh
    Inhibition of Nox2-activity reduces oxidative stress and Src kinase-mediated impaired autophagy ( a ) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalse. ( b ) Measurement of intracellular glutathione redox potential with Grx1-roGFP2. ( c ) Analysis of <t>Rac1</t> and ( d ) Src. ( e ) Immunoblot of precipitated p47 phox probed with an anti-phosphoserine or anti-p47 phox antibody. ( f ) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor. ( g ) Extracellular ROS production was assessed using Amplex-red dye. ( h ) Plasma membrane calcium influx was measured by analyzing the Fura-2 fluorescence quench rate upon addition of extracellular Mn 2+ . ( i ) Intracellular RNS generation was measured using DAF-FM. Bars represent average ±SEM from n=15 individual fibers for each condition in ( a , b , f , g , j and i ). Markers of autophagy were analyzed in isolated fibers (incubated with or without PP2) from FDBs. ( k ) Autophagosome formation was analyzed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. ( l ) Confocal microscopy detected p62-LC3 localization in single fibers from FDBs (scale bar=140 μm and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. <t>GAPDH</t> was detected as a loading control. Representative images are shown. Bars represent average ±SEM from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey’s post-hoc test. *p
    Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gapdh/product/Millipore
    Average 99 stars, based on 1300 article reviews
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    Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.

    Journal: Genes & Development

    Article Title: Foxp1 regulation of neonatal vocalizations via cortical development

    doi: 10.1101/gad.305037.117

    Figure Lengend Snippet: Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.

    Article Snippet: The following antibodies were used: mouse monoclonal anti-SUMO-1 (D-11) antibody (Santa Cruz Biotechnology, sc-5308), rabbit polyclonal anti-FOXP1 antibody , mouse monoclonal anti-FOXP1 (JC12) antibody (Abcam, ab32010), goat polyclonal anti-FOXP2 (N-16) antibody (Santa Cruz Biotechnology, sc-21068), mouse monoclonal anti-Flag M2 antibody (Sigma-Aldrich, F1804), mouse monoclonal anti-V5 antibody (Invitrogen, R960-25), goat polyclonal anti-GFP antibody (Rockland Immunochemicals, 600-101-215), chick polyclonal anti-GFP antibody (Aves Laboratories, GFP-1010), rabbit monoclonal anti-SUMO-2/3 (18H8) antibody (Cell Signaling Technology, 4971), rabbit polyclonal anti-PIAS2 antibody (Abcam, ab155556), rabbit polyclonal anti-PIAS3 (H-169) antibody (Santa Cruz Biotechnology, sc-14017), rabbit polyclonal anti-MAP2 antibody (Chemicon, AB5622), mouse monoclonal anti-CtBP (E-12) antibody (Santa Cruz Biotechnology, sc-17759), rabbit polyclonal anti-CDP (CUX1: M-222) antibody (Santa Cruz Biotechnology, sc-13024), rat anti-CTIP2 (Abcam, ab18465), rabbit polyclonal anti-HDAC1 antibody (Abcam, ab19845), mouse monoclonal anti-HDAC1 (10E2) antibody (Cell Signaling Technology, 5256), mouse monoclonal anti-HDAC2 (3F3) antibody (Cell Signaling Technology, 5113), rabbit monoclonal anti-MTA1 (D40D1) XP antibody (Cell Signaling Technology, 5647), rabbit polyclonal anti-MTA2 (H-170) antibody (Santa Cruz Biotechnology, sc-28731), rabbit polyclonal anti-p66β (GATAD2B) antibody (Novus Biologicals, NBP1-87358), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Millipore, MAB374), mouse (G3A1) mAb IgG1 isotype control (Cell Signaling Technology, 5415), normal rabbit IgG (Cell Signaling Technology, 2729), and normal goat IgG (Santa Cruz Biotechnology, sc-2028).

    Techniques: Western Blot, Immunoprecipitation

    Na v 1.1 levels are reduced in Scn1a RX/+ mice, and this reduction is not prevented by tau ablation. Levels of Na v 1.1 and total sodium channels (pan Na v ) in the parietal cortex of 8-month-old mice were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype). The average Na v 1.1 to pan Na v ratio in Scn1a +/+ / Tau +/+ mice was arbitrarily defined as 1.0. ***p

    Journal: Annals of Neurology

    Article Title: Tau Reduction Prevents Disease in a Mouse Model of Dravet Syndrome

    doi: 10.1002/ana.24230

    Figure Lengend Snippet: Na v 1.1 levels are reduced in Scn1a RX/+ mice, and this reduction is not prevented by tau ablation. Levels of Na v 1.1 and total sodium channels (pan Na v ) in the parietal cortex of 8-month-old mice were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype). The average Na v 1.1 to pan Na v ratio in Scn1a +/+ / Tau +/+ mice was arbitrarily defined as 1.0. ***p

    Article Snippet: After blocking for 1 hour in 5% bovine serum albumin diluted in Tris-buffered saline (BSA-TBS), membranes were incubated overnight at 4°C in anti-Nav 1.1 (1:1,000; Alomone Labs, Jerusalem, Israel), anti–pan-sodium channel (Pan Nav, 1:1,000; Sigma), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; Millipore, Billerica, MA), anti-tau clone Tau-5 (1:3,000; Life Technologies), anti-tau clone EP2456Y (1:1,000; Millipore), anti–phospho-tau Ser 396/404 clone PHF-1 (1:3,000, a gift from Dr P. Davies), anti–phospho-tau Thr231 clone CP9 (1:25, a gift from Dr P. Davies), or anti–phospho-PHF-tau pSer202+Thr205 clone AT8 (1:80; Thermo Scientific, Waltham, MA).

    Techniques: Mouse Assay, Western Blot

    Cortical levels of total and phosphorylated tau are not altered in Scn1a RX/+ mice. Levels of phospho-tau (PHF-1, Ser396/Ser404; AT8, Ser202/Thr205; CP9, Thr231) and total tau (Tau-5, EP2456Y) in the parietal cortex of 8-month-old mice of the indicated genotypes were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype) revealed no statistically significant differences between Scn1a RX/+ /Tau +/+ and Scn1a +/+ / Tau +/+ mice (Student t test). Average phospho-tau to EP2456Y ratios (PHF-1, AT8, CP9) or average total tau levels (Tau-5, EP2456Y) in Scn1a +/+ / Tau +/+ mice were arbitrarily defined as 1.0. Values represent mean ± standard error of the mean.

    Journal: Annals of Neurology

    Article Title: Tau Reduction Prevents Disease in a Mouse Model of Dravet Syndrome

    doi: 10.1002/ana.24230

    Figure Lengend Snippet: Cortical levels of total and phosphorylated tau are not altered in Scn1a RX/+ mice. Levels of phospho-tau (PHF-1, Ser396/Ser404; AT8, Ser202/Thr205; CP9, Thr231) and total tau (Tau-5, EP2456Y) in the parietal cortex of 8-month-old mice of the indicated genotypes were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype) revealed no statistically significant differences between Scn1a RX/+ /Tau +/+ and Scn1a +/+ / Tau +/+ mice (Student t test). Average phospho-tau to EP2456Y ratios (PHF-1, AT8, CP9) or average total tau levels (Tau-5, EP2456Y) in Scn1a +/+ / Tau +/+ mice were arbitrarily defined as 1.0. Values represent mean ± standard error of the mean.

    Article Snippet: After blocking for 1 hour in 5% bovine serum albumin diluted in Tris-buffered saline (BSA-TBS), membranes were incubated overnight at 4°C in anti-Nav 1.1 (1:1,000; Alomone Labs, Jerusalem, Israel), anti–pan-sodium channel (Pan Nav, 1:1,000; Sigma), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; Millipore, Billerica, MA), anti-tau clone Tau-5 (1:3,000; Life Technologies), anti-tau clone EP2456Y (1:1,000; Millipore), anti–phospho-tau Ser 396/404 clone PHF-1 (1:3,000, a gift from Dr P. Davies), anti–phospho-tau Thr231 clone CP9 (1:25, a gift from Dr P. Davies), or anti–phospho-PHF-tau pSer202+Thr205 clone AT8 (1:80; Thermo Scientific, Waltham, MA).

    Techniques: Mouse Assay, Western Blot

    Inhibition of Nox2-activity reduces oxidative stress and Src kinase-mediated impaired autophagy ( a ) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalse. ( b ) Measurement of intracellular glutathione redox potential with Grx1-roGFP2. ( c ) Analysis of Rac1 and ( d ) Src. ( e ) Immunoblot of precipitated p47 phox probed with an anti-phosphoserine or anti-p47 phox antibody. ( f ) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor. ( g ) Extracellular ROS production was assessed using Amplex-red dye. ( h ) Plasma membrane calcium influx was measured by analyzing the Fura-2 fluorescence quench rate upon addition of extracellular Mn 2+ . ( i ) Intracellular RNS generation was measured using DAF-FM. Bars represent average ±SEM from n=15 individual fibers for each condition in ( a , b , f , g , j and i ). Markers of autophagy were analyzed in isolated fibers (incubated with or without PP2) from FDBs. ( k ) Autophagosome formation was analyzed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. ( l ) Confocal microscopy detected p62-LC3 localization in single fibers from FDBs (scale bar=140 μm and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±SEM from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey’s post-hoc test. *p

    Journal: Nature communications

    Article Title: Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy

    doi: 10.1038/ncomms5425

    Figure Lengend Snippet: Inhibition of Nox2-activity reduces oxidative stress and Src kinase-mediated impaired autophagy ( a ) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalse. ( b ) Measurement of intracellular glutathione redox potential with Grx1-roGFP2. ( c ) Analysis of Rac1 and ( d ) Src. ( e ) Immunoblot of precipitated p47 phox probed with an anti-phosphoserine or anti-p47 phox antibody. ( f ) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor. ( g ) Extracellular ROS production was assessed using Amplex-red dye. ( h ) Plasma membrane calcium influx was measured by analyzing the Fura-2 fluorescence quench rate upon addition of extracellular Mn 2+ . ( i ) Intracellular RNS generation was measured using DAF-FM. Bars represent average ±SEM from n=15 individual fibers for each condition in ( a , b , f , g , j and i ). Markers of autophagy were analyzed in isolated fibers (incubated with or without PP2) from FDBs. ( k ) Autophagosome formation was analyzed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. ( l ) Confocal microscopy detected p62-LC3 localization in single fibers from FDBs (scale bar=140 μm and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±SEM from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey’s post-hoc test. *p

    Article Snippet: Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase), anti-active Rac1, anti-p47phox and anti-P-serine were purchased from Millipore.

    Techniques: Inhibition, Activity Assay, Fluorescence, Isolation, Incubation, Microscopy, Confocal Microscopy, Western Blot

    Neuronal specificity and efficacy of RNA interference with SUMO2/3 and its effects on survival of mouse primary cortical neurons. At days in vitro (DIV) 3, primary cortical neurons were transduced with lentiviral particles expressing EGFP as a reporter and either SUMO2/3 or control microRNA driven by the neuron-specific synapsin promoter. ( A ) Verification of transduction efficacy and neuronal specificity of cultures transduced with lentiviral particles driven by the synapsin promoter. Immunohistochemistry was performed after paraformaldehyde (PFA) fixation on DIV 12 with antibodies against EGFP, microtubuli-associated protein 2 (MAP2), and nuclear counterstain with DAPI. The multiplicity of infection (MOI) was ∼50. Scale bar=100 μ m. ( B ) Verification of knockdown efficiency of SUMO2/3 versus control microRNAs (1=LacZ, 2=non-targeting scrambled) with and without OGD (45 minutes and 3 hours reoxygenation) shown by a representative western blot analysis. Neuronal cultures were analyzed on DIV 12 and subjected to SDS-PAGE. Membranes were probed with antibodies against SUMO2/3, EGFP, and GAPDH. EGFP expression corresponded to an equal MOI of lentiviral particles and concomitant microRNA expression. GAPDH served as a housekeeping protein and equal loading control. ( C ) SUMO2/3 microRNA does not influence baseline survival over time up to DIV 12. In brief, microscopic pictures of EGFP fluorescence (indicative for microRNA delivery) were taken at DIV 6, 9, and 12 as described in the ‘Materials and methods' section. EGFP-expressing neurons were counted and ratios were calculated for DIV 9/6 (indicated in blue) and DIV 12/9 (red) to evaluate cell survival over time. We assumed an effect size > 0.15 and performed a prospective power analysis with α =0.05 and β =0.20. There was no significant difference between groups in a two-way repeated-measures ANOVA followed by Tukey's post hoc analysis. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP2, microtubule-associated protein 2; OGD, oxygen–glucose deprivation; SUMO2/3, small ubiquitin-like modifier-2/3.

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: SUMO2/3 conjugation is an endogenous neuroprotective mechanism

    doi: 10.1038/jcbfm.2011.112

    Figure Lengend Snippet: Neuronal specificity and efficacy of RNA interference with SUMO2/3 and its effects on survival of mouse primary cortical neurons. At days in vitro (DIV) 3, primary cortical neurons were transduced with lentiviral particles expressing EGFP as a reporter and either SUMO2/3 or control microRNA driven by the neuron-specific synapsin promoter. ( A ) Verification of transduction efficacy and neuronal specificity of cultures transduced with lentiviral particles driven by the synapsin promoter. Immunohistochemistry was performed after paraformaldehyde (PFA) fixation on DIV 12 with antibodies against EGFP, microtubuli-associated protein 2 (MAP2), and nuclear counterstain with DAPI. The multiplicity of infection (MOI) was ∼50. Scale bar=100 μ m. ( B ) Verification of knockdown efficiency of SUMO2/3 versus control microRNAs (1=LacZ, 2=non-targeting scrambled) with and without OGD (45 minutes and 3 hours reoxygenation) shown by a representative western blot analysis. Neuronal cultures were analyzed on DIV 12 and subjected to SDS-PAGE. Membranes were probed with antibodies against SUMO2/3, EGFP, and GAPDH. EGFP expression corresponded to an equal MOI of lentiviral particles and concomitant microRNA expression. GAPDH served as a housekeeping protein and equal loading control. ( C ) SUMO2/3 microRNA does not influence baseline survival over time up to DIV 12. In brief, microscopic pictures of EGFP fluorescence (indicative for microRNA delivery) were taken at DIV 6, 9, and 12 as described in the ‘Materials and methods' section. EGFP-expressing neurons were counted and ratios were calculated for DIV 9/6 (indicated in blue) and DIV 12/9 (red) to evaluate cell survival over time. We assumed an effect size > 0.15 and performed a prospective power analysis with α =0.05 and β =0.20. There was no significant difference between groups in a two-way repeated-measures ANOVA followed by Tukey's post hoc analysis. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP2, microtubule-associated protein 2; OGD, oxygen–glucose deprivation; SUMO2/3, small ubiquitin-like modifier-2/3.

    Article Snippet: Immunoblots Neurons were harvested as described previously ( ) and probed with a SUMO2/3-specific antibody (Invitrogen; 1:1,000), anti-GAPDH (anti-glyceraldehyde 3-phosphate dehydrogenase, Millipore; 1:75,000), and anti-EGFP (Santa Cruz Biotechnology, 1:1,000).

    Techniques: In Vitro, Transduction, Expressing, Immunohistochemistry, Infection, Western Blot, SDS Page, Fluorescence