Structured Review

Millipore anti β actin antibody
WT α-syn was degraded by the proteasome and the CMA pathway. PC12 cells stably overexpressing WT α-syn were treated with 10 μM MG132 ( A ) or 10 μM Lac ( B ) for 24 h. Cells were lysed and subjected to SDS-PAGE by using an anti-α-syn antibody. ( C ) Cells were treated with 10 μM MG132 or 10 μM Lac for 24 h and mRNA levels of WT α-syn were detected by using the RT-PCR. ( D ) Cells were labeled with a Lyso-Tracker for 20 min and was visualized by using a confocal microscopy. Representative images were shown. Scale bar: 10 mm. ( E ) Cells were treated with 100 μM CQ, 30 mg/mL HCQ, and 20 mM NH 4 Cl for 24 h. Cells were harvested and subjected to SDS-PAGE using an anti-α-syn antibody. ( F ) Cells were treated with 10 mM 3-MA and 20 mM NH 4 Cl for 24 h. α-Syn levels were determined by using Western blotting analysis. ( G ) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, 20 mM NH 4 Cl, 10 mM 3-MA, and a combination of NH 4 Cl with 3-MA for 24 h. RT-PCR was conducted to assess the mRNA levels of WT α-syn. ( H ) Lysates from PC12 cells were immuno-precipitated with an anti-α-syn antibody and blotted with an anti-LAMP2A antibody. IgG was used as a negative control. Representative blots of three independent experiments were shown. <t>β-Actin</t> levels demonstrated equal loading. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p
Anti β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "E46K Mutant α-Synuclein Is Degraded by Both Proteasome and Macroautophagy Pathway"

Article Title: E46K Mutant α-Synuclein Is Degraded by Both Proteasome and Macroautophagy Pathway

Journal: Molecules

doi: 10.3390/molecules23112839

WT α-syn was degraded by the proteasome and the CMA pathway. PC12 cells stably overexpressing WT α-syn were treated with 10 μM MG132 ( A ) or 10 μM Lac ( B ) for 24 h. Cells were lysed and subjected to SDS-PAGE by using an anti-α-syn antibody. ( C ) Cells were treated with 10 μM MG132 or 10 μM Lac for 24 h and mRNA levels of WT α-syn were detected by using the RT-PCR. ( D ) Cells were labeled with a Lyso-Tracker for 20 min and was visualized by using a confocal microscopy. Representative images were shown. Scale bar: 10 mm. ( E ) Cells were treated with 100 μM CQ, 30 mg/mL HCQ, and 20 mM NH 4 Cl for 24 h. Cells were harvested and subjected to SDS-PAGE using an anti-α-syn antibody. ( F ) Cells were treated with 10 mM 3-MA and 20 mM NH 4 Cl for 24 h. α-Syn levels were determined by using Western blotting analysis. ( G ) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, 20 mM NH 4 Cl, 10 mM 3-MA, and a combination of NH 4 Cl with 3-MA for 24 h. RT-PCR was conducted to assess the mRNA levels of WT α-syn. ( H ) Lysates from PC12 cells were immuno-precipitated with an anti-α-syn antibody and blotted with an anti-LAMP2A antibody. IgG was used as a negative control. Representative blots of three independent experiments were shown. β-Actin levels demonstrated equal loading. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p
Figure Legend Snippet: WT α-syn was degraded by the proteasome and the CMA pathway. PC12 cells stably overexpressing WT α-syn were treated with 10 μM MG132 ( A ) or 10 μM Lac ( B ) for 24 h. Cells were lysed and subjected to SDS-PAGE by using an anti-α-syn antibody. ( C ) Cells were treated with 10 μM MG132 or 10 μM Lac for 24 h and mRNA levels of WT α-syn were detected by using the RT-PCR. ( D ) Cells were labeled with a Lyso-Tracker for 20 min and was visualized by using a confocal microscopy. Representative images were shown. Scale bar: 10 mm. ( E ) Cells were treated with 100 μM CQ, 30 mg/mL HCQ, and 20 mM NH 4 Cl for 24 h. Cells were harvested and subjected to SDS-PAGE using an anti-α-syn antibody. ( F ) Cells were treated with 10 mM 3-MA and 20 mM NH 4 Cl for 24 h. α-Syn levels were determined by using Western blotting analysis. ( G ) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, 20 mM NH 4 Cl, 10 mM 3-MA, and a combination of NH 4 Cl with 3-MA for 24 h. RT-PCR was conducted to assess the mRNA levels of WT α-syn. ( H ) Lysates from PC12 cells were immuno-precipitated with an anti-α-syn antibody and blotted with an anti-LAMP2A antibody. IgG was used as a negative control. Representative blots of three independent experiments were shown. β-Actin levels demonstrated equal loading. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p

Techniques Used: Stable Transfection, SDS Page, Reverse Transcription Polymerase Chain Reaction, Labeling, Confocal Microscopy, Western Blot, Negative Control

The E46K mutant α-syn is degraded by a proteasome and a macro-autophagy pathway. ( A ) PC12 cells stably overexpressing the E46K mutant α-syn were treated with 10 μM MG132 for 24 h. Cells were lysed and subjected to SDS-PAGE using an anti-α-syn antibody. ( B ) Stable PC12 cells overexpressing E46K mutant α-syn were treated with 10 μM Lac for 24 h. α-Syn levels were assessed by using an anti-α-syn antibody. ( C ) Cells were treated with 10 μM MG132 or 10 μM Lac for 24 h and mRNA levels of the E46K mutant α-syn were detected by using RT-PCR. ( D ) Cells were labeled with Lyso-Tracker for 20 min and visualized by using a confocal microscopy. Representative images were shown. Scale bar: 10 mm. ( E ) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, and 20 mM NH 4 Cl for 24 h. Cells were lysed and subjected to SDS-PAGE using an anti-α-syn antibody. ( F ) Cells were incubated with 10 mM 3-MA, 20 mM NH 4 Cl, and a combination of 3-MA and NH 4 Cl for 24 h. Cells were lysed and subjected to immunoblot analysis using an anti-α-syn antibody. ( G ) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, 20 mM NH 4 Cl, 10 mM 3-MA, and a combination of NH 4 Cl with 3-MA for 24 h. RT-PCR was conducted to assess the mRNA levels of E46K mutant α-syn. ( H ) PC12 cells were treated with 100 mM Tre and 200 nM Rap for 24 h and α-syn levels were assessed using Western blotting. ( I ) PC12 cells were treated with Rap for indicated concentration and harvested for immunoblotting for α-syn. ( J ) PC12 cells were treated with 400 nM Rap for the indicated periods of time and cell extracts were subjected to the immunoblot. Representative blots of three independent experiments were shown. β-actin levels demonstrate equal loading. ( K ) Cells were treated with 100 mM Tre of 400 nM Rap for 24 h and mRNA levels of E46K mutant α-syn were detected by RT-PCR. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p
Figure Legend Snippet: The E46K mutant α-syn is degraded by a proteasome and a macro-autophagy pathway. ( A ) PC12 cells stably overexpressing the E46K mutant α-syn were treated with 10 μM MG132 for 24 h. Cells were lysed and subjected to SDS-PAGE using an anti-α-syn antibody. ( B ) Stable PC12 cells overexpressing E46K mutant α-syn were treated with 10 μM Lac for 24 h. α-Syn levels were assessed by using an anti-α-syn antibody. ( C ) Cells were treated with 10 μM MG132 or 10 μM Lac for 24 h and mRNA levels of the E46K mutant α-syn were detected by using RT-PCR. ( D ) Cells were labeled with Lyso-Tracker for 20 min and visualized by using a confocal microscopy. Representative images were shown. Scale bar: 10 mm. ( E ) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, and 20 mM NH 4 Cl for 24 h. Cells were lysed and subjected to SDS-PAGE using an anti-α-syn antibody. ( F ) Cells were incubated with 10 mM 3-MA, 20 mM NH 4 Cl, and a combination of 3-MA and NH 4 Cl for 24 h. Cells were lysed and subjected to immunoblot analysis using an anti-α-syn antibody. ( G ) Cells were treated with 100 μM CQ, 30 μg/mL HCQ, 20 mM NH 4 Cl, 10 mM 3-MA, and a combination of NH 4 Cl with 3-MA for 24 h. RT-PCR was conducted to assess the mRNA levels of E46K mutant α-syn. ( H ) PC12 cells were treated with 100 mM Tre and 200 nM Rap for 24 h and α-syn levels were assessed using Western blotting. ( I ) PC12 cells were treated with Rap for indicated concentration and harvested for immunoblotting for α-syn. ( J ) PC12 cells were treated with 400 nM Rap for the indicated periods of time and cell extracts were subjected to the immunoblot. Representative blots of three independent experiments were shown. β-actin levels demonstrate equal loading. ( K ) Cells were treated with 100 mM Tre of 400 nM Rap for 24 h and mRNA levels of E46K mutant α-syn were detected by RT-PCR. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p

Techniques Used: Mutagenesis, Stable Transfection, SDS Page, Reverse Transcription Polymerase Chain Reaction, Labeling, Confocal Microscopy, Incubation, Western Blot, Concentration Assay

E46K mutant α-syn turned over more slowly when compared with WT α-syn. ( A ) PC12 cells stably overexpressing WT and E46K mutant α-syn were treated with 10 mg/mL CHX for the indicated periods of time and α-syn levels were measured by Western blotting. ( B ) Representative blots of three independent experiments were shown. β-Actin levels demonstrated equal loading. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p
Figure Legend Snippet: E46K mutant α-syn turned over more slowly when compared with WT α-syn. ( A ) PC12 cells stably overexpressing WT and E46K mutant α-syn were treated with 10 mg/mL CHX for the indicated periods of time and α-syn levels were measured by Western blotting. ( B ) Representative blots of three independent experiments were shown. β-Actin levels demonstrated equal loading. Data were expressed as means ± SD for three independent experiments performed in triplicate. * p

Techniques Used: Mutagenesis, Stable Transfection, Western Blot

2) Product Images from "Connexin 30 deficiency attenuates A2 astrocyte responses and induces severe neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride Parkinson’s disease animal model"

Article Title: Connexin 30 deficiency attenuates A2 astrocyte responses and induces severe neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride Parkinson’s disease animal model

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-018-1251-0

Reduced MPTP-induced GFAP upregulation in Cx30-deficient mice. a , b GFAP immunostaining of the striatum on day 1 ( a ) and 7 ( b ) after injection of WT and Cx30 KO mice with normal saline (NS) or MPTP. c Quantification of Western blot analysis of GFAP protein levels normalised to β-actin levels. d , e GFAP immunostaining ( d ) and GFAP-positive cell numbers ( e ) in the SNc in WT and Cx30 KO mice 7 days after injection of NS or MPTP. Data are the mean ± SEM of n = 3 ( c ) or 4 ( e ) mice. * p
Figure Legend Snippet: Reduced MPTP-induced GFAP upregulation in Cx30-deficient mice. a , b GFAP immunostaining of the striatum on day 1 ( a ) and 7 ( b ) after injection of WT and Cx30 KO mice with normal saline (NS) or MPTP. c Quantification of Western blot analysis of GFAP protein levels normalised to β-actin levels. d , e GFAP immunostaining ( d ) and GFAP-positive cell numbers ( e ) in the SNc in WT and Cx30 KO mice 7 days after injection of NS or MPTP. Data are the mean ± SEM of n = 3 ( c ) or 4 ( e ) mice. * p

Techniques Used: Mouse Assay, Immunostaining, Injection, Western Blot

Effects of MPTP on Cx30 levels in the striatum of WT mice on days 1 and 7 after injection of normal saline (NS) or MPTP. a Number of Cx30-immunoreactive dots counted by Nikon NIS-Element software. b qRT-PCR analysis of Cx30 mRNA levels normalised to Gapdh mRNA. c Cx30 protein levels measured by Western blotting and normalised to β-actin levels. Data are the mean ± SEM of n = 4–5 mice per group. * p
Figure Legend Snippet: Effects of MPTP on Cx30 levels in the striatum of WT mice on days 1 and 7 after injection of normal saline (NS) or MPTP. a Number of Cx30-immunoreactive dots counted by Nikon NIS-Element software. b qRT-PCR analysis of Cx30 mRNA levels normalised to Gapdh mRNA. c Cx30 protein levels measured by Western blotting and normalised to β-actin levels. Data are the mean ± SEM of n = 4–5 mice per group. * p

Techniques Used: Mouse Assay, Injection, Software, Quantitative RT-PCR, Western Blot

Effects of MPTP on Cx43 expression in the striatum of WT and Cx30 KO mice on days 1 and 7 after the treatment with normal saline (NS) or MPTP. a , b Number of Cx43-immunoreactive dots counted by Nikon NIS-Element software. c , d , qRT-PCR analysis of Cx30 mRNA levels normalised to Gapdh mRNA. e , f Cx30 protein levels measured by Western blotting and normalised to β-actin levels. Analyses were performed on day 1 ( a , c , e ) and day 7 ( b , d , f ) after treatment. Data are presented as the mean ± SEM of n = 4–5 mice per group. * p
Figure Legend Snippet: Effects of MPTP on Cx43 expression in the striatum of WT and Cx30 KO mice on days 1 and 7 after the treatment with normal saline (NS) or MPTP. a , b Number of Cx43-immunoreactive dots counted by Nikon NIS-Element software. c , d , qRT-PCR analysis of Cx30 mRNA levels normalised to Gapdh mRNA. e , f Cx30 protein levels measured by Western blotting and normalised to β-actin levels. Analyses were performed on day 1 ( a , c , e ) and day 7 ( b , d , f ) after treatment. Data are presented as the mean ± SEM of n = 4–5 mice per group. * p

Techniques Used: Expressing, Mouse Assay, Software, Quantitative RT-PCR, Western Blot

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Article Title: Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages
Article Snippet: Supernatants from these lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P PVDF transfer membranes. .. The membranes were then incubated with the following primary antibodies: anti-ApoE (EP1347Y; Abcam), anti-apolipoprotein B (AB742; EMD Millipore), anti-HIV-1 p24 rabbit polyclonal antibody (65–004; Bioacademia, Japan), anti-HIV-1 p24 mouse monoclonal antibody (A2-851-100; Icosagen, Estonia), anti-HIV-1 Env (KD-247) [ ], anti-VSV Glycoprotein (P5D4; Sigma), anti-HA (HA-7; Sigma), and anti- β -actin (AC-15; Sigma). .. After the incubation with HRP-labeled secondary antibodies, proteins were visualized using Western Lightning Plus-ECL (PerkinElmer) and an Amersham Imager 600 imaging system (GE Healthcare).

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    Millipore mouse anti β actin
    K562 cells with ectopic HD5 expression. (A) Screening of cell lines for endogenous HD5 expression. HD5 mRNA levels were measured in total cellular RNA by qRT-PCR and expressed as percentages of <t>β-actin</t> (Act B) mRNA levels. (B) HD5 mRNA expression
    Mouse Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin/product/Millipore
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    K562 cells with ectopic HD5 expression. (A) Screening of cell lines for endogenous HD5 expression. HD5 mRNA levels were measured in total cellular RNA by qRT-PCR and expressed as percentages of β-actin (Act B) mRNA levels. (B) HD5 mRNA expression

    Journal: Journal of Virology

    Article Title: Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy

    doi: 10.1128/JVI.02030-16

    Figure Lengend Snippet: K562 cells with ectopic HD5 expression. (A) Screening of cell lines for endogenous HD5 expression. HD5 mRNA levels were measured in total cellular RNA by qRT-PCR and expressed as percentages of β-actin (Act B) mRNA levels. (B) HD5 mRNA expression

    Article Snippet: After blots were stripped with Restore Western blot striping buffer (Thermo Scientific, Waltham, MA), they were incubated with mouse anti-β-actin (Sigma, St. Louis, MO) (1:5,000).

    Techniques: Expressing, Quantitative RT-PCR, Activated Clotting Time Assay

    ShRNA plasmid design and in vitro LGI1-shRNA downregulation. A) shRNA construct: LGI1-shRNA is under the promoter mU6 (murine U6 small-non-coding-RNA), meanwhile the EGFP (enhanced green fluorescent protein) is under the independent promoter PGK (phosphoglycerate kinase). NeoR/KanR (Neomycin/Kanamycin Resistance). B) LGI1 expression in neuronal cultures. The first lane is the endogenous level of LGI1 in untreated primary cultures, the second lane shows the levels of LGI1 in cultures transduced with scramble virus and the fourth and last lane shows the levels of LGI1 in cultures transduced with shRNA-LGI1. The β-actin and EGFP signals are also shown. EGFP intensity was detected to evaluate that the reporter gene was also expressed at the same time as the shRNA and hence could be a reliable source of transduction rate. C) Quantification of LGI1 concentrations detected by the western blot method. p=0.005 two-tailed Student’s t-test (blots n=5 from 5 distinct preparations).

    Journal: bioRxiv

    Article Title: LGI1 downregulation increases neuronal circuit excitability

    doi: 10.1101/2019.12.17.879833

    Figure Lengend Snippet: ShRNA plasmid design and in vitro LGI1-shRNA downregulation. A) shRNA construct: LGI1-shRNA is under the promoter mU6 (murine U6 small-non-coding-RNA), meanwhile the EGFP (enhanced green fluorescent protein) is under the independent promoter PGK (phosphoglycerate kinase). NeoR/KanR (Neomycin/Kanamycin Resistance). B) LGI1 expression in neuronal cultures. The first lane is the endogenous level of LGI1 in untreated primary cultures, the second lane shows the levels of LGI1 in cultures transduced with scramble virus and the fourth and last lane shows the levels of LGI1 in cultures transduced with shRNA-LGI1. The β-actin and EGFP signals are also shown. EGFP intensity was detected to evaluate that the reporter gene was also expressed at the same time as the shRNA and hence could be a reliable source of transduction rate. C) Quantification of LGI1 concentrations detected by the western blot method. p=0.005 two-tailed Student’s t-test (blots n=5 from 5 distinct preparations).

    Article Snippet: The following antibodies and dilutions were used: 1:250 polyclonal goat-anti-LGI1 (Santa Cruz, c-19), 1:4000 monoclonal mouse-anti β-actin (Sigma), 1:1000 rabbit-anti-GFP (Abcam ab6556).

    Techniques: shRNA, Plasmid Preparation, In Vitro, Construct, Expressing, Transduction, Western Blot, Two Tailed Test

    Effects of various concentrations of the resveratrol on KGN cells. Cells were incubated with resveratrol at 0, 10, 25, 50, and 100 μmol/L for 24 h. A, SIRT1 mRNA levels were assessed by real‐time PCR and calculated after normalization to EF1α mRNA levels. B, The protein levels of SIRT1 were quantified by Western blotting, and β‐actin was used as the control. C, The protein levels were quantified using ImageJ. D, VEGF protein levels were analyzed by ELISA. E, The mtDNA copy number was determined using real‐time PCR. Fold differences are shown compared with the control, for which the value was defined as 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Journal: Reproductive Medicine and Biology

    Article Title: Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia, et al. Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia

    doi: 10.1002/rmb2.12323

    Figure Lengend Snippet: Effects of various concentrations of the resveratrol on KGN cells. Cells were incubated with resveratrol at 0, 10, 25, 50, and 100 μmol/L for 24 h. A, SIRT1 mRNA levels were assessed by real‐time PCR and calculated after normalization to EF1α mRNA levels. B, The protein levels of SIRT1 were quantified by Western blotting, and β‐actin was used as the control. C, The protein levels were quantified using ImageJ. D, VEGF protein levels were analyzed by ELISA. E, The mtDNA copy number was determined using real‐time PCR. Fold differences are shown compared with the control, for which the value was defined as 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Article Snippet: Blots were then incubated for 1 hour at room temperature (25°C) with rabbit monoclonal SIRT1 antibody (1:400; Santa Cruz Biotechnology, Inc), rabbit monoclonal HIF‐1a antibody (1:1000; Epitomics), or mouse monoclonal β‐actin antibody (1:5000; Sigma‐Aldrich) as the primary antibody, and anti‐rabbit immunoglobulin IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) or anti‐mouse IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) as the secondary antibody.

    Techniques: Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    Protective effects of resveratrol against CoCl 2 ‐induced hypoxic stress. KGN cells were cultured in medium containing 100 μmol/L CoCl 2 with or without the 50 μmol/L resveratrol. A, SIRT1 mRNA levels were assessed by real‐time PCR and calculated after normalization to EF1α mRNA levels. B, The protein levels of SIRT1 were quantified by Western blotting, and β‐actin was used as the control protein. C, The protein levels were quantified using ImageJ. D, VEGF protein levels were analyzed by ELISA. E, The mtDNA copy number was determined using real‐time PCR. Fold differences are shown compared with the control, for which the value was defined as 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Journal: Reproductive Medicine and Biology

    Article Title: Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia, et al. Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia

    doi: 10.1002/rmb2.12323

    Figure Lengend Snippet: Protective effects of resveratrol against CoCl 2 ‐induced hypoxic stress. KGN cells were cultured in medium containing 100 μmol/L CoCl 2 with or without the 50 μmol/L resveratrol. A, SIRT1 mRNA levels were assessed by real‐time PCR and calculated after normalization to EF1α mRNA levels. B, The protein levels of SIRT1 were quantified by Western blotting, and β‐actin was used as the control protein. C, The protein levels were quantified using ImageJ. D, VEGF protein levels were analyzed by ELISA. E, The mtDNA copy number was determined using real‐time PCR. Fold differences are shown compared with the control, for which the value was defined as 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Article Snippet: Blots were then incubated for 1 hour at room temperature (25°C) with rabbit monoclonal SIRT1 antibody (1:400; Santa Cruz Biotechnology, Inc), rabbit monoclonal HIF‐1a antibody (1:1000; Epitomics), or mouse monoclonal β‐actin antibody (1:5000; Sigma‐Aldrich) as the primary antibody, and anti‐rabbit immunoglobulin IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) or anti‐mouse IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) as the secondary antibody.

    Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    Effects of CoCl 2 ‐induced hypoxic stress on mRNA expression and protein secretion. KGN cells were incubated for 24 h in medium containing with 10 μmol/L or 100 μmol/L CoCl 2 . A, SIRT1 mRNA levels were assessed by real‐time PCR and calculated after normalization to EF1α mRNA levels. B, The protein levels of SIRT1 were quantified by Western blotting, and β‐actin was used as the control. C, The protein levels were quantified using ImageJ. D, VEGF protein levels were analyzed by ELISA. E, The mtDNA copy number was determined using real‐time PCR. Fold differences are shown compared with the control, for which the value was defined as 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Journal: Reproductive Medicine and Biology

    Article Title: Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia, et al. Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia

    doi: 10.1002/rmb2.12323

    Figure Lengend Snippet: Effects of CoCl 2 ‐induced hypoxic stress on mRNA expression and protein secretion. KGN cells were incubated for 24 h in medium containing with 10 μmol/L or 100 μmol/L CoCl 2 . A, SIRT1 mRNA levels were assessed by real‐time PCR and calculated after normalization to EF1α mRNA levels. B, The protein levels of SIRT1 were quantified by Western blotting, and β‐actin was used as the control. C, The protein levels were quantified using ImageJ. D, VEGF protein levels were analyzed by ELISA. E, The mtDNA copy number was determined using real‐time PCR. Fold differences are shown compared with the control, for which the value was defined as 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Article Snippet: Blots were then incubated for 1 hour at room temperature (25°C) with rabbit monoclonal SIRT1 antibody (1:400; Santa Cruz Biotechnology, Inc), rabbit monoclonal HIF‐1a antibody (1:1000; Epitomics), or mouse monoclonal β‐actin antibody (1:5000; Sigma‐Aldrich) as the primary antibody, and anti‐rabbit immunoglobulin IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) or anti‐mouse IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) as the secondary antibody.

    Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    Effect of resveratrol on HIF‐1α. KGN cells were incubated for 24 h in medium containing 100 μmol/L CoCl 2 with or without 50 μmol/L resveratrol (n = 3). A, The expression of HIF‐1α was quantified by Western blotting. The levels of HIF‐1α were normalized to levels of β‐actin (n = 3). B, The protein levels were quantified using ImageJ. C, VEGF protein levels were analyzed by ELISA. Fold differences are shown compared with the control, for which the value was defined 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Journal: Reproductive Medicine and Biology

    Article Title: Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia, et al. Resveratrol protects mitochondrial quantity by activating SIRT1/PGC‐1α expression during ovarian hypoxia

    doi: 10.1002/rmb2.12323

    Figure Lengend Snippet: Effect of resveratrol on HIF‐1α. KGN cells were incubated for 24 h in medium containing 100 μmol/L CoCl 2 with or without 50 μmol/L resveratrol (n = 3). A, The expression of HIF‐1α was quantified by Western blotting. The levels of HIF‐1α were normalized to levels of β‐actin (n = 3). B, The protein levels were quantified using ImageJ. C, VEGF protein levels were analyzed by ELISA. Fold differences are shown compared with the control, for which the value was defined 1.0. The data are presented as the mean ± SEM, n = 3. Statistically significant differences are indicated by brackets: * P

    Article Snippet: Blots were then incubated for 1 hour at room temperature (25°C) with rabbit monoclonal SIRT1 antibody (1:400; Santa Cruz Biotechnology, Inc), rabbit monoclonal HIF‐1a antibody (1:1000; Epitomics), or mouse monoclonal β‐actin antibody (1:5000; Sigma‐Aldrich) as the primary antibody, and anti‐rabbit immunoglobulin IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) or anti‐mouse IgG peroxidase‐labeled secondary antibody (1:10 000; GE Healthcare Life Science) as the secondary antibody.

    Techniques: Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    Temporal changes in synaptic plasticity-related signals in the mouse hippocampus following methotrexate (MTX) administration. (A) N-methyl-D-aspartic acid receptor 1 (NMDAR1). (B) Calcium/calmodulin-dependent protein kinase II (CaMKII). (C) Extracellular signal-regulated kinase 1/2 (ERK1/2). (D) cAMP responsive element-binding protein (CREB). (E) Glutamate receptor 1 (GluR1). The relative expression levels of phosphorylated (P) and total (T) forms were determined by densitometry and normalized to β-actin signals. Furthermore, to examine the activation levels of each signal, the relative levels of the P forms were normalized to their T-forms. Data are reported as mean ± SEM of three mice per time point. Values for protein expression levels from the hippocampus of vehicle-treated controls were arbitrarily defined as 1 (bar graphs). a P

    Journal: Neural Regeneration Research

    Article Title: Temporal profiles of synaptic plasticity-related signals in adult mouse hippocampus with methotrexate treatment ☆

    doi: 10.3969/j.issn.1673-5374.2012.21.008

    Figure Lengend Snippet: Temporal changes in synaptic plasticity-related signals in the mouse hippocampus following methotrexate (MTX) administration. (A) N-methyl-D-aspartic acid receptor 1 (NMDAR1). (B) Calcium/calmodulin-dependent protein kinase II (CaMKII). (C) Extracellular signal-regulated kinase 1/2 (ERK1/2). (D) cAMP responsive element-binding protein (CREB). (E) Glutamate receptor 1 (GluR1). The relative expression levels of phosphorylated (P) and total (T) forms were determined by densitometry and normalized to β-actin signals. Furthermore, to examine the activation levels of each signal, the relative levels of the P forms were normalized to their T-forms. Data are reported as mean ± SEM of three mice per time point. Values for protein expression levels from the hippocampus of vehicle-treated controls were arbitrarily defined as 1 (bar graphs). a P

    Article Snippet: Membranes were stripped and re-probed with a mouse monoclonal anti-β-actin (1:20 000; Sigma-Aldrich) for normalization.

    Techniques: Binding Assay, Expressing, Activation Assay, Mouse Assay

    Time-response changes of neurotrophic factors in the mouse hippocampus following administration of methotrexate (MTX). (A) Brain-derived neurotrophic factor (BDNF). (B) Glial cell line-derived neurotrophic factor (GDNF). The relative expression levels of proteins were determined by densitometry and normalized to β-actin signals. Data are reported as mean ± SEM. n = 3 mice per time point. Values for protein expression levels from the hippocampus of vehicle-treated controls (Cont) were arbitrarily defined as 1 (bar graphs). a P

    Journal: Neural Regeneration Research

    Article Title: Temporal profiles of synaptic plasticity-related signals in adult mouse hippocampus with methotrexate treatment ☆

    doi: 10.3969/j.issn.1673-5374.2012.21.008

    Figure Lengend Snippet: Time-response changes of neurotrophic factors in the mouse hippocampus following administration of methotrexate (MTX). (A) Brain-derived neurotrophic factor (BDNF). (B) Glial cell line-derived neurotrophic factor (GDNF). The relative expression levels of proteins were determined by densitometry and normalized to β-actin signals. Data are reported as mean ± SEM. n = 3 mice per time point. Values for protein expression levels from the hippocampus of vehicle-treated controls (Cont) were arbitrarily defined as 1 (bar graphs). a P

    Article Snippet: Membranes were stripped and re-probed with a mouse monoclonal anti-β-actin (1:20 000; Sigma-Aldrich) for normalization.

    Techniques: Derivative Assay, Expressing, Mouse Assay