anti-actin antibody Santa Cruz Biotechnology Search Results


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  • 95
    Millipore anti actb
    Activation of EGFR tyrosine kinase regulates the binding of <t>BCL2</t> to BECN1 in hypoxia. (A) BCL2 protein level over a 72 h time course in hypoxia was determined by western blot in U87 and A549 cells. <t>ACTB</t> was used as a loading control. (B) BCL2
    Anti Actb, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Santa Cruz Biotechnology santa cruz biotechnology actin
    In vivo validation of autophagy inhibition upon Atg5 downregulation. (a) ATG5 shows by western blot a downregulation across a range of tissue in adult mice treated with dox for 6 weeks, except the brain, which displays no alterations in ATG5 in whole tissue extracts. <t>ACTB</t> serves as a loading control in all tissues except for heart and muscle for which total <t>actin</t> was used instead. (b) ATG5i mice display an increase in SQSTM1 in the indicated tissues via IHC. (c) Atg5 downregulation is associated with the development of large proteinaceous aggregates in the liver (yellow arrows). Additionally, cellular degeneration of the exocrine and endocrine pancreas is visible by H E analysis (yellow asterisk). Scale bars: 100 μm in b and c.
    Santa Cruz Biotechnology Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 2360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Santa Cruz Biotechnology anti beta actin sc 2778 santa cruz biotechnology
    In vivo validation of autophagy inhibition upon Atg5 downregulation. (a) ATG5 shows by western blot a downregulation across a range of tissue in adult mice treated with dox for 6 weeks, except the brain, which displays no alterations in ATG5 in whole tissue extracts. <t>ACTB</t> serves as a loading control in all tissues except for heart and muscle for which total <t>actin</t> was used instead. (b) ATG5i mice display an increase in SQSTM1 in the indicated tissues via IHC. (c) Atg5 downregulation is associated with the development of large proteinaceous aggregates in the liver (yellow arrows). Additionally, cellular degeneration of the exocrine and endocrine pancreas is visible by H E analysis (yellow asterisk). Scale bars: 100 μm in b and c.
    Anti Beta Actin Sc 2778 Santa Cruz Biotechnology, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Santa Cruz Biotechnology β actin ab
    IFN-α down-regulates telomerase activity in the cytoplasm and the nucleus of CD8 + T cells. A representative gel showing telomerase activity (TRAP Assay, see Material and Methods) of cytoplasmic and nuclear extracts from 5 × 10 3 viable CD8 + T cells tested 72 hours following activation with anti CD3 Ab plus rhIL-2 is shown in A. Graph showing pooled results of the effect of IFN-α on telomerase activity of fractioned extracts from 3 separate donors is shown in B. A representative experiment showing the effect of IFN-α on hTERT expression, tested on cytoplasmic and nuclear extracts from 5×10 6 viable CD8 + T lymphocytes 36 hours following activation by Western Blot is shown in C. Quality of extracts was tested using anti <t>β</t> actin and Histone H1 Ab respectively. The graph in the panel D shows the mean ± SD of the ratio protein/β-actin and Histone H1 for cytoplasmic and nuclear compartments respectively, for 3 donors. The effect of IFN-α on the distribution and composition of NPCs was tested on CD8 + ). In panel E a representative image of the staining is shown. The images were collected on an inverted confocal microscope (Olympus) with a 60X objective (N.A. 1.45) with a SIM scanner using the Fluoview software. The fluorescence within a region of interest surrounding the nuclear envelope in twelve mid-plane sections was measured. All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p
    β Actin Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Santa Cruz Biotechnology ab against β actin
    SAM inhibits the LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with SAM (0.5 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. <t>β-Actin</t> was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.
    Ab Against β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 83/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Santa Cruz Biotechnology anti actin polyclonal ab i 19
    SAM inhibits the LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with SAM (0.5 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. <t>β-Actin</t> was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.
    Anti Actin Polyclonal Ab I 19, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Santa Cruz Biotechnology anti actin isoform antibodies
    SAM inhibits the LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with SAM (0.5 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. <t>β-Actin</t> was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.
    Anti Actin Isoform Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Santa Cruz Biotechnology anti actin western analysis
    SAM inhibits the LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with SAM (0.5 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. <t>β-Actin</t> was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.
    Anti Actin Western Analysis, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Santa Cruz Biotechnology anti actin goat polyclonal ab
    SAM inhibits the LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with SAM (0.5 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. <t>β-Actin</t> was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.
    Anti Actin Goat Polyclonal Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc β actin antibody
    Detection of PMCA4a in reproductive luminal fluids and its acquisition on caudal sperm A ) Representative Western blot of FLFs collected during pro-estrus and estrus and metestrus and diestrus (40 µg proteins loaded). The ~128 kDa PMCA4a is seen in pro-estrus and estrus and is marginally present at metestrus and diestrus. Caudal epididymal luminal fluid was used as a positive control. The membrane was stripped and re-probed for HSC70 as a loading control. B ) Western blots of VLF, ULF, and OLF recovered after superovulation demonstrate the presence of the ~128 kDa PMCA4a. Sperm protein was used as a positive control. The membrane was stripped and re-probed for <t>β-actin</t> as a loading control. C ) Quantitation of Western blot data shown in B; the relative expression was determined using VLF as 1. The data represent the mean (±SEM) of a minimum of three independent experiments, and the intensity was quantified by Image J software. ANOVA and t -tests were performed on the mean and P values were calculated. * P = 0.03 indicates a significantly increase amount of PMCA4a in OLF compared to that in VLF. D ) A peak shift of fluorescence intensity to the right, indicates increase amounts of PMCA4a in sperm incubated in FLF compared to PBS for 2 h and treated as described in Materials and Methods.
    β Actin Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1585 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Santa Cruz Biotechnology rabbit polyclonal anti actin ab
    Detection of PMCA4a in reproductive luminal fluids and its acquisition on caudal sperm A ) Representative Western blot of FLFs collected during pro-estrus and estrus and metestrus and diestrus (40 µg proteins loaded). The ~128 kDa PMCA4a is seen in pro-estrus and estrus and is marginally present at metestrus and diestrus. Caudal epididymal luminal fluid was used as a positive control. The membrane was stripped and re-probed for HSC70 as a loading control. B ) Western blots of VLF, ULF, and OLF recovered after superovulation demonstrate the presence of the ~128 kDa PMCA4a. Sperm protein was used as a positive control. The membrane was stripped and re-probed for <t>β-actin</t> as a loading control. C ) Quantitation of Western blot data shown in B; the relative expression was determined using VLF as 1. The data represent the mean (±SEM) of a minimum of three independent experiments, and the intensity was quantified by Image J software. ANOVA and t -tests were performed on the mean and P values were calculated. * P = 0.03 indicates a significantly increase amount of PMCA4a in OLF compared to that in VLF. D ) A peak shift of fluorescence intensity to the right, indicates increase amounts of PMCA4a in sperm incubated in FLF compared to PBS for 2 h and treated as described in Materials and Methods.
    Rabbit Polyclonal Anti Actin Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Santa Cruz Biotechnology rabbit anti actin primary ab
    Detection of PMCA4a in reproductive luminal fluids and its acquisition on caudal sperm A ) Representative Western blot of FLFs collected during pro-estrus and estrus and metestrus and diestrus (40 µg proteins loaded). The ~128 kDa PMCA4a is seen in pro-estrus and estrus and is marginally present at metestrus and diestrus. Caudal epididymal luminal fluid was used as a positive control. The membrane was stripped and re-probed for HSC70 as a loading control. B ) Western blots of VLF, ULF, and OLF recovered after superovulation demonstrate the presence of the ~128 kDa PMCA4a. Sperm protein was used as a positive control. The membrane was stripped and re-probed for <t>β-actin</t> as a loading control. C ) Quantitation of Western blot data shown in B; the relative expression was determined using VLF as 1. The data represent the mean (±SEM) of a minimum of three independent experiments, and the intensity was quantified by Image J software. ANOVA and t -tests were performed on the mean and P values were calculated. * P = 0.03 indicates a significantly increase amount of PMCA4a in OLF compared to that in VLF. D ) A peak shift of fluorescence intensity to the right, indicates increase amounts of PMCA4a in sperm incubated in FLF compared to PBS for 2 h and treated as described in Materials and Methods.
    Rabbit Anti Actin Primary Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Activation of EGFR tyrosine kinase regulates the binding of BCL2 to BECN1 in hypoxia. (A) BCL2 protein level over a 72 h time course in hypoxia was determined by western blot in U87 and A549 cells. ACTB was used as a loading control. (B) BCL2

    Journal: Autophagy

    Article Title: Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia

    doi: 10.1080/15548627.2016.1164357

    Figure Lengend Snippet: Activation of EGFR tyrosine kinase regulates the binding of BCL2 to BECN1 in hypoxia. (A) BCL2 protein level over a 72 h time course in hypoxia was determined by western blot in U87 and A549 cells. ACTB was used as a loading control. (B) BCL2

    Article Snippet: Primary antibodies: anti-ATG5 (2630), anti-BECN1 (3738), anti-EGFR (2232) and anti-phospho-EGFR (Y1068) (2234) were purchased from Cell Signaling Technology, anti-LC3B (0231-100/LC3-5F10) from NanoTools, anti-CAV1 (sc-894) and anti-BCL2 (sc-7382) from Santa Cruz Biotechnology, and anti-ACTB from Sigma-Aldrich (A3853).

    Techniques: Activation Assay, Binding Assay, Western Blot

    In vivo validation of autophagy inhibition upon Atg5 downregulation. (a) ATG5 shows by western blot a downregulation across a range of tissue in adult mice treated with dox for 6 weeks, except the brain, which displays no alterations in ATG5 in whole tissue extracts. ACTB serves as a loading control in all tissues except for heart and muscle for which total actin was used instead. (b) ATG5i mice display an increase in SQSTM1 in the indicated tissues via IHC. (c) Atg5 downregulation is associated with the development of large proteinaceous aggregates in the liver (yellow arrows). Additionally, cellular degeneration of the exocrine and endocrine pancreas is visible by H E analysis (yellow asterisk). Scale bars: 100 μm in b and c.

    Journal: Autophagy

    Article Title: A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo

    doi: 10.1080/15548627.2018.1458172

    Figure Lengend Snippet: In vivo validation of autophagy inhibition upon Atg5 downregulation. (a) ATG5 shows by western blot a downregulation across a range of tissue in adult mice treated with dox for 6 weeks, except the brain, which displays no alterations in ATG5 in whole tissue extracts. ACTB serves as a loading control in all tissues except for heart and muscle for which total actin was used instead. (b) ATG5i mice display an increase in SQSTM1 in the indicated tissues via IHC. (c) Atg5 downregulation is associated with the development of large proteinaceous aggregates in the liver (yellow arrows). Additionally, cellular degeneration of the exocrine and endocrine pancreas is visible by H E analysis (yellow asterisk). Scale bars: 100 μm in b and c.

    Article Snippet: Anti-ATG5 (Abcam, ab108327; 1:1000), anti-LC3 (Nanotools, Clone 5F10; 1:1000), anti-ACTB (Sigma, A5441; 1:10,000), anti-ACTIN (Santa Cruz Biotechnology, I-19; 1:5000 [no longer commercially available]), anti-ACTA2/α-SMA (Abcam, ab5694; 1:1000), anti-COL1A1 (Abcam, ab34710; 1:2000), anti-poly UBIQUITIN (Enzo, Clone FK1; 1:5000), anti-turboGFP (Pierce, PA5-22,688; 1:2000), anti-rabbit HRP and anti-mouse HRP (GE Healthcare, NA934V and NA931V; 1:5000)

    Techniques: In Vivo, Inhibition, Western Blot, Mouse Assay, Immunohistochemistry

    MGO‐induced c‐FLIP L down‐regulation was mediated by the suppression of p65 expression. ( A ) EA.hy26 cells were treated with the indicated concentrations of MGO for 18 hrs. Equal amounts of cell lysates (40 μg) were electrophoresed and Western blotted as described above. ( B ) The cells were treated with the indicated concentrations of MGO for 18 hrs. The total RNA was isolated, and RT‐PCR was performed, as described in Materials and methods . A representative study is shown; two additional experiments yielded similar results. ( C ) EA.hy926 cells were treated with MGO in the presence or absence of CHX for the indicated times (upper panel). NAC plus CHX in the absence or presence of MGO (upper panel). Western blotting was performed with anti‐p65 and anti‐actin antibody (actin served as the loading control). ( D ) After transient transfection with the empty vector or p65 expression vector, the cells were treated with MGO for 18 hrs. Apoptosis was assessed by determining the proportion of cells in the sub‐G1 fraction by FACS. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Methylglyoxal‐induced apoptosis is dependent on the suppression of c‐FLIPL expression via down‐regulation of p65 in endothelial cells

    doi: 10.1111/jcmm.13188

    Figure Lengend Snippet: MGO‐induced c‐FLIP L down‐regulation was mediated by the suppression of p65 expression. ( A ) EA.hy26 cells were treated with the indicated concentrations of MGO for 18 hrs. Equal amounts of cell lysates (40 μg) were electrophoresed and Western blotted as described above. ( B ) The cells were treated with the indicated concentrations of MGO for 18 hrs. The total RNA was isolated, and RT‐PCR was performed, as described in Materials and methods . A representative study is shown; two additional experiments yielded similar results. ( C ) EA.hy926 cells were treated with MGO in the presence or absence of CHX for the indicated times (upper panel). NAC plus CHX in the absence or presence of MGO (upper panel). Western blotting was performed with anti‐p65 and anti‐actin antibody (actin served as the loading control). ( D ) After transient transfection with the empty vector or p65 expression vector, the cells were treated with MGO for 18 hrs. Apoptosis was assessed by determining the proportion of cells in the sub‐G1 fraction by FACS. * P

    Article Snippet: The anti‐actin antibody was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, FACS

    Apoptotic protein levels. Cleaved caspase 3 ELISA and Western blot results for wildtype mice and β2-adrenergic receptor KO mice for pro-apoptotic proteins (top panel-cleaved caspase 3, Bax, Cytochrome C) and anti-apoptotic proteins (bottom panel-phosphorylated Akt, Bcl-xL). A representative Western blot is provided. All Western blot data were normalized to beta actin levels. ELISA data was normalized to protein loaded into well. *P

    Journal: PLoS ONE

    Article Title: ?2-Adrenergic Receptor Knockout Mice Exhibit A Diabetic Retinopathy Phenotype

    doi: 10.1371/journal.pone.0070555

    Figure Lengend Snippet: Apoptotic protein levels. Cleaved caspase 3 ELISA and Western blot results for wildtype mice and β2-adrenergic receptor KO mice for pro-apoptotic proteins (top panel-cleaved caspase 3, Bax, Cytochrome C) and anti-apoptotic proteins (bottom panel-phosphorylated Akt, Bcl-xL). A representative Western blot is provided. All Western blot data were normalized to beta actin levels. ELISA data was normalized to protein loaded into well. *P

    Article Snippet: Primary antibodies used were phosphorylated Akt (Serine 473), Akt, Cytochrome C, Bax, Bcl-xL, SOCS3, GFAP, phosphorylated insulin receptor (tyrosine 1150/1151), insulin receptor (all purchased from Cell Signaling, Danvers, MA), and beta actin (Santa Cruz).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Mouse Assay

    Insulin Receptor Levels and localization. Left Panel: Western blot results for phosphorylated insulin receptor (Tyr 1150/1151) in β2-adrenergic receptor knockout mice vs. wildtype. Western blot data were normalized to beta actin. N = 5 mice in each group. *P

    Journal: PLoS ONE

    Article Title: ?2-Adrenergic Receptor Knockout Mice Exhibit A Diabetic Retinopathy Phenotype

    doi: 10.1371/journal.pone.0070555

    Figure Lengend Snippet: Insulin Receptor Levels and localization. Left Panel: Western blot results for phosphorylated insulin receptor (Tyr 1150/1151) in β2-adrenergic receptor knockout mice vs. wildtype. Western blot data were normalized to beta actin. N = 5 mice in each group. *P

    Article Snippet: Primary antibodies used were phosphorylated Akt (Serine 473), Akt, Cytochrome C, Bax, Bcl-xL, SOCS3, GFAP, phosphorylated insulin receptor (tyrosine 1150/1151), insulin receptor (all purchased from Cell Signaling, Danvers, MA), and beta actin (Santa Cruz).

    Techniques: Western Blot, Knock-Out, Mouse Assay

    Tumorigenic potential of low passage mucoepidermoid carcinoma cells sorted for ALDH/CD44 A., B. Graphs depicting tumor volume of A. UM-HMC-3A or B. UM-HMC-3B xenograft cells FACS-sorted for ALDH/CD44. Scaffolds were seeded with either 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells and transplanted into the subcutaneous space of SCID mice. Existing tumors were retrieved, re-sorted and 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells seeded into new scaffolds, and serially passaged in vivo . C. Table depicting the number of tumors grown in the ALDH high CD44 high versus ALDH low CD44 low populations for each passage performed. D. H E staining of tumors generated with FACS-sorted ALDH high CD44 high and ALDH low CD44 low cells. Images were taken at 100X. E. UM-HMC-3A and UM-HMC-3B cells were sorted for ALDH high CD44 high or combined ALDH high CD44 low , ALDH low CD44 high , and ALDH low CD44 low (non-CSC population). NP-40 lysis buffer was used to prepare whole cell lysates that were resolved using PAGE. Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K, p-EGFR; 1:2000 dilution of EGFR, and beta-actin.

    Journal: Oncotarget

    Article Title: ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas

    doi:

    Figure Lengend Snippet: Tumorigenic potential of low passage mucoepidermoid carcinoma cells sorted for ALDH/CD44 A., B. Graphs depicting tumor volume of A. UM-HMC-3A or B. UM-HMC-3B xenograft cells FACS-sorted for ALDH/CD44. Scaffolds were seeded with either 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells and transplanted into the subcutaneous space of SCID mice. Existing tumors were retrieved, re-sorted and 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells seeded into new scaffolds, and serially passaged in vivo . C. Table depicting the number of tumors grown in the ALDH high CD44 high versus ALDH low CD44 low populations for each passage performed. D. H E staining of tumors generated with FACS-sorted ALDH high CD44 high and ALDH low CD44 low cells. Images were taken at 100X. E. UM-HMC-3A and UM-HMC-3B cells were sorted for ALDH high CD44 high or combined ALDH high CD44 low , ALDH low CD44 high , and ALDH low CD44 low (non-CSC population). NP-40 lysis buffer was used to prepare whole cell lysates that were resolved using PAGE. Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K, p-EGFR; 1:2000 dilution of EGFR, and beta-actin.

    Article Snippet: Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K (Cell Signaling; Beverly, MA, USA); 1:2000 dilution of EGFR, a 1:1000 dilution of p-EGFR, and beta-actin (Santa Cruz Biotechnology; Santa Cruz, CA, USA) overnight at 4°C.

    Techniques: FACS, Mouse Assay, In Vivo, Staining, Generated, Lysis, Polyacrylamide Gel Electrophoresis

    Induction of apoptosis by cisplatin is independent of PUMA mechanism. ( A , B ) The QBC939 and FRH 0201 cells were exposed to various cisplatin concentrations for 72 h. The PUMA and E-cadherin protein expression levels in the cell lysate were examined by Western blot analysis using anti-PUMA (E-cadherin) antibody, and the antibodies against ß-actin which served as an internal control. ( C , D ) The QBC939 and FRH 0201 cells were exposed to 20 μg/mL cisplatin for the indicated times. The PUMA and E-cadherin protein expression level was measured by Western blot. ( E , F ) QBC939 and FRH0201 cells (1 × 10 6 /mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, PUMA protein expression level was measured by Western blot. ( G , H ) QBC939 and FRH0201 cells (1 × 10 6 /mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, after which time the percentage of apoptotic cells was determined by flow cytometric analysis, as described in “Materials and Methods”. Cisplatin combined with PUMA siRNA did not induce obvious apoptosis in QBC939 and FRH 0201 cells ( * P

    Journal: International Journal of Molecular Sciences

    Article Title: RNA Interference Targeting Slug Increases Cholangiocarcinoma Cell Sensitivity to Cisplatin via Upregulating PUMA

    doi: 10.3390/ijms12010385

    Figure Lengend Snippet: Induction of apoptosis by cisplatin is independent of PUMA mechanism. ( A , B ) The QBC939 and FRH 0201 cells were exposed to various cisplatin concentrations for 72 h. The PUMA and E-cadherin protein expression levels in the cell lysate were examined by Western blot analysis using anti-PUMA (E-cadherin) antibody, and the antibodies against ß-actin which served as an internal control. ( C , D ) The QBC939 and FRH 0201 cells were exposed to 20 μg/mL cisplatin for the indicated times. The PUMA and E-cadherin protein expression level was measured by Western blot. ( E , F ) QBC939 and FRH0201 cells (1 × 10 6 /mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, PUMA protein expression level was measured by Western blot. ( G , H ) QBC939 and FRH0201 cells (1 × 10 6 /mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, after which time the percentage of apoptotic cells was determined by flow cytometric analysis, as described in “Materials and Methods”. Cisplatin combined with PUMA siRNA did not induce obvious apoptosis in QBC939 and FRH 0201 cells ( * P

    Article Snippet: Primary antibodies were as follows: Anti-Slug (1:200 dilution), Anti–PUMA (1:400 dilution), and Anti-ß-actin (1:500 dilution), all from Santa Cruz Biotechnology.

    Techniques: Expressing, Western Blot, Flow Cytometry

    TBS-soluble tau levels in rTg4510 mice. (A, D) Representative western blot images for tau and β-actin in the cerebral cortex (A) and the hippocampus (D). Left and right bands in each group correspond to rTg4510_TxC mice and rTg4510_CxT mice, respectively. (B, C, E, F) Densitometric analysis of the levels of tau and AT8-positive phosphorylated tau in the cerebral cortex (B, C) and the hippocampus (E, F) in rTg4510 mice fed with control chow diet (n = 8) and Shiga-Y5 (SY5)-containing chow diet (n = 7). Data are the mean ± standard error of the mean. Significance (Mann Whitney test): *p

    Journal: PLoS ONE

    Article Title: Study of tau pathology in male rTg4510 mice fed with a curcumin derivative Shiga-Y5

    doi: 10.1371/journal.pone.0208440

    Figure Lengend Snippet: TBS-soluble tau levels in rTg4510 mice. (A, D) Representative western blot images for tau and β-actin in the cerebral cortex (A) and the hippocampus (D). Left and right bands in each group correspond to rTg4510_TxC mice and rTg4510_CxT mice, respectively. (B, C, E, F) Densitometric analysis of the levels of tau and AT8-positive phosphorylated tau in the cerebral cortex (B, C) and the hippocampus (E, F) in rTg4510 mice fed with control chow diet (n = 8) and Shiga-Y5 (SY5)-containing chow diet (n = 7). Data are the mean ± standard error of the mean. Significance (Mann Whitney test): *p

    Article Snippet: The membranes were blocked with 5% non-fat milk in Tris-buffered saline (25 mM Tris-HCl, pH 7.4, 0.9% NaCl) containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature, incubated overnight with primary antibodies including mouse monoclonal antibodies against phosphorylated tau (Ser202 and Thr205) (clone AT8; 1:2000; Thermo Fisher Scientific, Waltham, MA, USA) and β-actin (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit polyclonal anti-tau antibody (1:1000000; DAKO, Glostrup, Denmark) at 4°C, and further incubated with horseradish peroxidase-conjugated goat polyclonal antibody against mouse IgG (1:20000; Jackson ImmunoResearch, West Grove, PA, USA) or rabbit IgG (1:20000; Jackson ImmunoResearch) for 1 h. Immunoreactive proteins were visualized with chemiluminescence (SuperSignal West Pico; Thermo Fisher Scientific, Waltham, MA, USA) using a lumino-image analyzer (LAS-4000mini; Fujifilm, Tokyo, Japan).

    Techniques: Mouse Assay, Western Blot, MANN-WHITNEY

    CB 2  receptor is expressed in mouse penis. Immunostaining and western blotting analysis was used to detect CB 2  receptor on penis from wild type and ApoE −/−  mice. (a) CB 2  was strongly colocalized with  α -actin (smooth muscle cells marker) as well as PECAM (endothelial cell marker) in the corpus cavernosum and dorsal vessels of wild type and ApoE −/−  mice. The graph shows representative images obtained from 8 different animals. (b) CB 2  in the mouse penis was confirmed by western blotting which revealed a specific single band at the expected molecular weight (approximately 45 kDa). The graph shows a representative gel of western blotting showing the expression of CB 2  in penis from wild type and ApoE −/−  mice from 4 independent experiments.

    Journal: Clinical and Developmental Immunology

    Article Title: Treatment with CB2 Agonist JWH-133 Reduces Histological Features Associated with Erectile Dysfunction in Hypercholesterolemic Mice

    doi: 10.1155/2013/263846

    Figure Lengend Snippet: CB 2 receptor is expressed in mouse penis. Immunostaining and western blotting analysis was used to detect CB 2 receptor on penis from wild type and ApoE −/− mice. (a) CB 2 was strongly colocalized with α -actin (smooth muscle cells marker) as well as PECAM (endothelial cell marker) in the corpus cavernosum and dorsal vessels of wild type and ApoE −/− mice. The graph shows representative images obtained from 8 different animals. (b) CB 2 in the mouse penis was confirmed by western blotting which revealed a specific single band at the expected molecular weight (approximately 45 kDa). The graph shows a representative gel of western blotting showing the expression of CB 2 in penis from wild type and ApoE −/− mice from 4 independent experiments.

    Article Snippet: Immunostaining in ApoE−/− Mouse Penis Six μ m cryosections from mice penes were fixed in acetone at room temperature and immunostained with the following antibodies: anti-CB2 (1 : 100, cat number sc-25494, Santa Cruz Biotechnology, Inc.) or anti-CB2 (1 : 100, cat number 301550, Cayman Chemical, Inc.), anti-α -actin (1 : 100, cat number sc-32251, Santa Cruz Biotechnology, Inc.), anti-PECAM (1 : 100, cat number sc-1506, Santa Cruz Biotechnology, Inc.), anti-rabbit IgG conjugated with Alexa Fluor-555 secondary antibody (1 : 400, cat number A31572, Invitrogen, Inc.), anti-mouse IgG conjugated with Alexa Fluor-647 secondary antibody (1 : 400, cat number A31571, Invitrogen, Inc.), and anti-goat IgG conjugated with Alexa Fluor-488 secondary antibody (1 : 400, cat number A21467, Invitrogen, Inc.).

    Techniques: Immunostaining, Western Blot, Mouse Assay, Marker, Molecular Weight, Expressing

    Inhibition of hypertrophy in right ventricular (RV) cardiomyocytes by dietary vitamin D supplementation in PH rats. (A) and (B), Representative images of hearts from rats in each experimental group. (A), Wheat germ agglutinin (WGA), α-actin, and 4’,6-diamidino-2-phenylindole (DAPI) staining of RV cross sections. Scale bars, 20 μm. (B) Hematoxylin and eosin (H E) staining. Above panels show magnified images of cardiomyocytes in RV wall. Scale bars, 20 μm (above panels) and 1 mm (below panels). (C) Quantification of RV cardiomyocyte with cross-sectional area stained (n = 5 rats per group). Data are mean ± SEM. * P

    Journal: PLoS ONE

    Article Title: Therapeutic impact of dietary vitamin D supplementation for preventing right ventricular remodeling and improving survival in pulmonary hypertension

    doi: 10.1371/journal.pone.0180615

    Figure Lengend Snippet: Inhibition of hypertrophy in right ventricular (RV) cardiomyocytes by dietary vitamin D supplementation in PH rats. (A) and (B), Representative images of hearts from rats in each experimental group. (A), Wheat germ agglutinin (WGA), α-actin, and 4’,6-diamidino-2-phenylindole (DAPI) staining of RV cross sections. Scale bars, 20 μm. (B) Hematoxylin and eosin (H E) staining. Above panels show magnified images of cardiomyocytes in RV wall. Scale bars, 20 μm (above panels) and 1 mm (below panels). (C) Quantification of RV cardiomyocyte with cross-sectional area stained (n = 5 rats per group). Data are mean ± SEM. * P

    Article Snippet: To quantify the cross-sectional area of RV cardiomyocytes, immunofluorescence staining of short-axis heart sections was performed with primary antibodies to α-actin (1:500, Santa Cruz) and Wheat Germ Agglutinin conjugated with Alexa Fluor 647 (1:100, Invitrogen).

    Techniques: Inhibition, Whole Genome Amplification, Staining

    Effects of DHT and DL3 on hsp70 and hsp90 expression. (A) LNCaP cells were incubated for 48 h in phenol-free medium supplemented with 10% charcoal-dextran stripped FBS (SFBS), then treated for 24 h with 0-10 nM of DHT in the absence or presence of 20 μ M of DL3. Cell lysates were analyzed by immunoblotting with antibodies to AR, PSA, hsp70-1 and hsp90 with ß-actin as a loading control. (B) LNCaP cells were treated as described in (A). Total RNA was extracted and expression of AR, PSA, hsp70-1 with ß-actin as a loading control was determined by qRT-PCR and presented as ratios to ß-actin. *p

    Journal: International journal of oncology

    Article Title: Regulation of heat shock protein 70-1 expression by androgen receptor and its signaling in human prostate cancer cells

    doi:

    Figure Lengend Snippet: Effects of DHT and DL3 on hsp70 and hsp90 expression. (A) LNCaP cells were incubated for 48 h in phenol-free medium supplemented with 10% charcoal-dextran stripped FBS (SFBS), then treated for 24 h with 0-10 nM of DHT in the absence or presence of 20 μ M of DL3. Cell lysates were analyzed by immunoblotting with antibodies to AR, PSA, hsp70-1 and hsp90 with ß-actin as a loading control. (B) LNCaP cells were treated as described in (A). Total RNA was extracted and expression of AR, PSA, hsp70-1 with ß-actin as a loading control was determined by qRT-PCR and presented as ratios to ß-actin. *p

    Article Snippet: The cells were washed in cold PBS/5 mM EDTA, scraped into a lysis buffer, and analyzed by Western blotting ( ) using antibodies against PSA (DakoCytomation, Glostrup, Denmark), AR (Epitomics, Burlingame, CA), hsp70-1 (Cell Signaling Technology, Inc., Danvers, MA), hsp90 (Santa Cruz Biotechnology, Santa Cruz, CA), and ß-actin (Santa Cruz).

    Techniques: Expressing, Incubation, Quantitative RT-PCR

    Mechanistic study on DL3-induced hsp70-1 downregulation. (A) LNCaP cells in medium supplemented with 10% FBS were treated for 16 h with 20 μ M of DL3 and/or 5 μ g/ml of MG132. (B) LNCaP cells in medium supplemented with 10% SFBS were treated for 24 h with 20 μ M of DL3, 1 nM of DHT, and/or 5 μ g/ml of CHX. (C) LNCaP cells were treated for different times with 5 μ g/ml of ActD in the absence or presence of 20 μ M of DL3. Total RNA was extracted and analyzed by qRT-PCR. (D) LNCaP cells, incubated in medium or treated with 20 μ M of DL3, were lyzed and mixed with the same volume of 0.6 M isolation sucrose buffer. Cell nucleus pellets obtained were incubated in transcription reaction buffer. Total RNA in the reaction mixture was extracted, and hsp70-1 and ß-actin mRNA was assessed by real-time RT-PCR. Samples without in vitro transcription reaction were used as controls (not shown). (E) LNCaP cells, treated for 4 h with 20 μ M of DL3 or Bic, were fixed in 1% formaldehyde. After washing and incubation in glycine, cells were scrapped into PBS supplemented with 5 mM PMSF, pelleted, resuspended in SDS lysis buffer, and sonicated to sheer DNA. AR-chromatin-transcription factor complex was precipitated with antibodies to AR with IgG from normal rabbit as a negative control. With 1:150 dilution of total DNA as a positive control (total input), DNA obtained from the immunoprecipitation was analyzed by using qPCR. (F) A portion of the ChIP samples was analyzed by immunoblotting to evaluate AR levels. In a parallel setting, total cell lysates were precipitated with the same AR antibody to evaluate the total cellular AR level in each treatment group.

    Journal: International journal of oncology

    Article Title: Regulation of heat shock protein 70-1 expression by androgen receptor and its signaling in human prostate cancer cells

    doi:

    Figure Lengend Snippet: Mechanistic study on DL3-induced hsp70-1 downregulation. (A) LNCaP cells in medium supplemented with 10% FBS were treated for 16 h with 20 μ M of DL3 and/or 5 μ g/ml of MG132. (B) LNCaP cells in medium supplemented with 10% SFBS were treated for 24 h with 20 μ M of DL3, 1 nM of DHT, and/or 5 μ g/ml of CHX. (C) LNCaP cells were treated for different times with 5 μ g/ml of ActD in the absence or presence of 20 μ M of DL3. Total RNA was extracted and analyzed by qRT-PCR. (D) LNCaP cells, incubated in medium or treated with 20 μ M of DL3, were lyzed and mixed with the same volume of 0.6 M isolation sucrose buffer. Cell nucleus pellets obtained were incubated in transcription reaction buffer. Total RNA in the reaction mixture was extracted, and hsp70-1 and ß-actin mRNA was assessed by real-time RT-PCR. Samples without in vitro transcription reaction were used as controls (not shown). (E) LNCaP cells, treated for 4 h with 20 μ M of DL3 or Bic, were fixed in 1% formaldehyde. After washing and incubation in glycine, cells were scrapped into PBS supplemented with 5 mM PMSF, pelleted, resuspended in SDS lysis buffer, and sonicated to sheer DNA. AR-chromatin-transcription factor complex was precipitated with antibodies to AR with IgG from normal rabbit as a negative control. With 1:150 dilution of total DNA as a positive control (total input), DNA obtained from the immunoprecipitation was analyzed by using qPCR. (F) A portion of the ChIP samples was analyzed by immunoblotting to evaluate AR levels. In a parallel setting, total cell lysates were precipitated with the same AR antibody to evaluate the total cellular AR level in each treatment group.

    Article Snippet: The cells were washed in cold PBS/5 mM EDTA, scraped into a lysis buffer, and analyzed by Western blotting ( ) using antibodies against PSA (DakoCytomation, Glostrup, Denmark), AR (Epitomics, Burlingame, CA), hsp70-1 (Cell Signaling Technology, Inc., Danvers, MA), hsp90 (Santa Cruz Biotechnology, Santa Cruz, CA), and ß-actin (Santa Cruz).

    Techniques: Quantitative RT-PCR, Incubation, Isolation, In Vitro, Lysis, Sonication, Negative Control, Positive Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Increased GAP-43 protein expression upon hnRNP-Q1 knockdown. (A) GAP-43 and γ-actin protein levels were assessed by immunoblot in N2a cell lysates 72 h after hnRNP-Q1 #1, hnRNP-Q1 #2, hnRNP-Q1 #3, or Scr siRNA transfection. n = 6, one-way analysis of variance (ANOVA), Dunnett’s posthoc, hnRNP-R p values: Scr vs. Q1 #1, p = 0.3897; Scr vs. Q1 #2, p = 0.2057; Scr vs. Q1 #3, p = 0.1801; hnRNP-Q3 p values: Scr vs. Q1 #1, p = 0.8869; Scr vs. Q1 #2, p = 0.4025; Scr vs. Q1 #3, p = 0.8486; hnRNP-Q1 p values: Scr vs. Q1 #1, p

    Journal: Molecular Biology of the Cell

    Article Title: hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translationGAP43, MARCKS, and CAP23 modulate PI(4,5)P

    doi: 10.1091/mbc.E15-07-0504

    Figure Lengend Snippet: Increased GAP-43 protein expression upon hnRNP-Q1 knockdown. (A) GAP-43 and γ-actin protein levels were assessed by immunoblot in N2a cell lysates 72 h after hnRNP-Q1 #1, hnRNP-Q1 #2, hnRNP-Q1 #3, or Scr siRNA transfection. n = 6, one-way analysis of variance (ANOVA), Dunnett’s posthoc, hnRNP-R p values: Scr vs. Q1 #1, p = 0.3897; Scr vs. Q1 #2, p = 0.2057; Scr vs. Q1 #3, p = 0.1801; hnRNP-Q3 p values: Scr vs. Q1 #1, p = 0.8869; Scr vs. Q1 #2, p = 0.4025; Scr vs. Q1 #3, p = 0.8486; hnRNP-Q1 p values: Scr vs. Q1 #1, p

    Article Snippet: Antibodies, immunoblotting, and immunofluorescence The following antibodies were used for immunoblotting: hnRNP-Q/R (1:1000; Sigma-Aldrich), GAP-43 (1:5000; Abcam, Cambridge, MA), γ-actin (1:10,000; Santa Cruz, Dallas, TX), α-tubulin (1:50,000; Sigma-Aldrich), IRDye 680LT donkey anti-mouse (1:20,000; Li-Cor, Lincoln, NE), IRDye 800CW donkey anti-mouse (1:20,000; Li-Cor), and IRDye 800CW donkey anti-rabbit (1:20,000, Li-Cor).

    Techniques: Expressing, Transfection

    hnRNP-Q1 directly binds a Gap-43 5′-UTR GQ sequence through the RGG box. (A) Flag-tagged hnRNP-Q1 was immunoprecipitated from N2a cell lysates, and copurified endogenous mRNAs were assessed by qRT-PCR. n = 3, one-way ANOVA, Dunnett’s posthoc, p values: Gapdh , p = 0.9276; Gap-43 , p = 0.0002. (B) Biotinylated RNA probes corresponding to FL Gap-43 mRNA, specific Gap-43 mRNA sequences, and/or deletions, or the γ-actin 3′-UTR were in vitro transcribed (see C and D for RNA probe purity). The (C) subregion or (D) FL RNA probes were incubated with recombinant GST or GST-hnRNP-Q1 protein and precipitated with NeutrAvidin beads. Copurified protein was assessed by GST immunoblot. Relative band intensity is listed below the immunoblots, and RNA probe integrity is shown by formaldehyde gel electrophoresis. (E) Representative fluorescence spectroscopy binding curve of the hnRNP-Q1 RGG box peptide and 2AP-labeled Gap-43 5′GQ RNA probe complex in 150 mM KCl and in the presence of a fivefold excess of the hepatitis C virus core peptide. The K d value determined from triplicate experiments was 131 ± 14 nM.

    Journal: Molecular Biology of the Cell

    Article Title: hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translationGAP43, MARCKS, and CAP23 modulate PI(4,5)P

    doi: 10.1091/mbc.E15-07-0504

    Figure Lengend Snippet: hnRNP-Q1 directly binds a Gap-43 5′-UTR GQ sequence through the RGG box. (A) Flag-tagged hnRNP-Q1 was immunoprecipitated from N2a cell lysates, and copurified endogenous mRNAs were assessed by qRT-PCR. n = 3, one-way ANOVA, Dunnett’s posthoc, p values: Gapdh , p = 0.9276; Gap-43 , p = 0.0002. (B) Biotinylated RNA probes corresponding to FL Gap-43 mRNA, specific Gap-43 mRNA sequences, and/or deletions, or the γ-actin 3′-UTR were in vitro transcribed (see C and D for RNA probe purity). The (C) subregion or (D) FL RNA probes were incubated with recombinant GST or GST-hnRNP-Q1 protein and precipitated with NeutrAvidin beads. Copurified protein was assessed by GST immunoblot. Relative band intensity is listed below the immunoblots, and RNA probe integrity is shown by formaldehyde gel electrophoresis. (E) Representative fluorescence spectroscopy binding curve of the hnRNP-Q1 RGG box peptide and 2AP-labeled Gap-43 5′GQ RNA probe complex in 150 mM KCl and in the presence of a fivefold excess of the hepatitis C virus core peptide. The K d value determined from triplicate experiments was 131 ± 14 nM.

    Article Snippet: Antibodies, immunoblotting, and immunofluorescence The following antibodies were used for immunoblotting: hnRNP-Q/R (1:1000; Sigma-Aldrich), GAP-43 (1:5000; Abcam, Cambridge, MA), γ-actin (1:10,000; Santa Cruz, Dallas, TX), α-tubulin (1:50,000; Sigma-Aldrich), IRDye 680LT donkey anti-mouse (1:20,000; Li-Cor, Lincoln, NE), IRDye 800CW donkey anti-mouse (1:20,000; Li-Cor), and IRDye 800CW donkey anti-rabbit (1:20,000, Li-Cor).

    Techniques: Sequencing, Immunoprecipitation, Quantitative RT-PCR, In Vitro, Incubation, Recombinant, Western Blot, Nucleic Acid Electrophoresis, Fluorescence, Spectroscopy, Binding Assay, Labeling

    B cell infection by DENV promotes MAPK phosphorylation. B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1). The cells were harvested after 2h, 24h or 48h p.i., and the expression of ERK, p38 and JNK MAPK were analyzed in the cell lysates by western blotting, using the indicated antibodies. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding non phosphorylated one; β actin staining were performed as a loading control and is shown in the bottom of the figure. Data are representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    doi: 10.1371/journal.pone.0143391

    Figure Lengend Snippet: B cell infection by DENV promotes MAPK phosphorylation. B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1). The cells were harvested after 2h, 24h or 48h p.i., and the expression of ERK, p38 and JNK MAPK were analyzed in the cell lysates by western blotting, using the indicated antibodies. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding non phosphorylated one; β actin staining were performed as a loading control and is shown in the bottom of the figure. Data are representative of three independent experiments.

    Article Snippet: The membranes were incubated with anti-phospho-p44/42 MAPK (Erk1/2) (1:2000), anti-phospho-p38 MAPK (1:1000), anti-phospho-SAPK/JNK (1:1000), anti-p44/42 MAPK (Erk1/2) (1:5000), anti-p38 MAPK (1:5000), anti-JNK (1:1000) (Cell Signaling Technology, Beverly, MA), anti-phosphotyrosine (4G10 clone; Merck Millipore, MA), anti-phospho AKT (1:200, Santa Cruz Biotechnology, Dallas, TX), anti-AKT (1:2000; Cell Signaling Technology), and anti-and β-actin (1:2000, Santa Cruz Biotechnology).

    Techniques: Infection, Cell Culture, Expressing, Western Blot, Staining

    FAO inhibition shows MYC-dependent bioenergetic and growth effects in vivo . ( a ) Immunoblot analysis of indicated protein expression in TN and RP patient-derived xenografts and human non-tumor reduction mammoplasty tissues. ( b ) Fold change in metabolite levels in etomoxir-treated xenografts versus vehicle-treated tumors. Values are shown as min-to-max box plots from three mice in each group. ( c ) Etomoxir- and vehicle-treated tumors were examined by immunoblotting for indicated protein expression. pAMPK/AMPK ratio was normalized to β-actin. ( d ) FVB/N mice with orthotopic MTB-TOM tumor allografts were treated with vehicle or etomoxir (40 mg/kg) daily for 14 d. Growth plots are shown. ( e ) Left, NOD/SCID mice with orthotopic HCI-002 xenografts were treated with vehicle or etomoxir (40 or 60 mg/kg) daily for 21 d. Growth plots are shown. Statistical analysis was performed using a log-rank test. ( f ) NOD/SCID mice with orthotopic HCI-009 xenografts were treated with vehicle or etomoxir (40 mg/kg) daily for 21 d. Growth plots are shown. ( g ) Left, representative Ki-67 and TUNEL staining of untreated, 40 or 60 mg/kg etomoxir-treated HCI-002 tumors from mice euthanized at the end of the study. Right, quantification of percent Ki-67 positive cells per field and number of TUNEL positive cells per field. Number of mice analyzed in each treatment is indicated. Scale bar indicates 200 µm. All differential metabolite abundance analyses were performed using the limma R package. A two-tailed unpaired t -test was used to compare experimental groups ( c–g ). Values shown are mean ± s.e.m. from three individual mice ( c ), six mice in the control group and seven mice in the experimental group ( d ), seven mice in the control and 40 mg/kg etomoxir groups and five mice in the 60 mg/kg group ( e ), three mice in each group ( f ), or three high-powered (20×) fields from two separate areas of each tumor ( g ). ^ P ≤ 0.10, * P ≤ 0.05, ** P

    Journal: Nature medicine

    Article Title: Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer

    doi: 10.1038/nm.4055

    Figure Lengend Snippet: FAO inhibition shows MYC-dependent bioenergetic and growth effects in vivo . ( a ) Immunoblot analysis of indicated protein expression in TN and RP patient-derived xenografts and human non-tumor reduction mammoplasty tissues. ( b ) Fold change in metabolite levels in etomoxir-treated xenografts versus vehicle-treated tumors. Values are shown as min-to-max box plots from three mice in each group. ( c ) Etomoxir- and vehicle-treated tumors were examined by immunoblotting for indicated protein expression. pAMPK/AMPK ratio was normalized to β-actin. ( d ) FVB/N mice with orthotopic MTB-TOM tumor allografts were treated with vehicle or etomoxir (40 mg/kg) daily for 14 d. Growth plots are shown. ( e ) Left, NOD/SCID mice with orthotopic HCI-002 xenografts were treated with vehicle or etomoxir (40 or 60 mg/kg) daily for 21 d. Growth plots are shown. Statistical analysis was performed using a log-rank test. ( f ) NOD/SCID mice with orthotopic HCI-009 xenografts were treated with vehicle or etomoxir (40 mg/kg) daily for 21 d. Growth plots are shown. ( g ) Left, representative Ki-67 and TUNEL staining of untreated, 40 or 60 mg/kg etomoxir-treated HCI-002 tumors from mice euthanized at the end of the study. Right, quantification of percent Ki-67 positive cells per field and number of TUNEL positive cells per field. Number of mice analyzed in each treatment is indicated. Scale bar indicates 200 µm. All differential metabolite abundance analyses were performed using the limma R package. A two-tailed unpaired t -test was used to compare experimental groups ( c–g ). Values shown are mean ± s.e.m. from three individual mice ( c ), six mice in the control group and seven mice in the experimental group ( d ), seven mice in the control and 40 mg/kg etomoxir groups and five mice in the 60 mg/kg group ( e ), three mice in each group ( f ), or three high-powered (20×) fields from two separate areas of each tumor ( g ). ^ P ≤ 0.10, * P ≤ 0.05, ** P

    Article Snippet: The primary antibodies used are as follows: β-Actin (Actin) (sc-47778 HRP, Santa Cruz, 1:10,000), PGC1α (ab54481, Abcam, 1:500), BBOX1 (WH0008424M1, Sigma Aldrich, 1:500), CPT2 (ab71435, Abcam, 1:500), FASN (SAB1403807, Sigma Aldrich, 1:1000), pACC1/2 (11818, Cell Signaling, 1:1000), ACC1 (4190, Cell Signaling, 1:1000), ACC2 (8578, Cell Signaling, 1:1000), AMPK (2532, Cell Signaling, 1:1000), pAMPK (2535, Cell Signaling, 1:1000) and c-MYC (MYC) (ab32072, Abcam, 1:1000).

    Techniques: Inhibition, In Vivo, Expressing, Derivative Assay, Mouse Assay, TUNEL Assay, Staining, Two Tailed Test

    Alterations in the expression of proteins involved in the metabolism [figure-6 (i) a b], oxidative stress [figure-6 (ii) a b], and cell death [figure-6 (iii) a b] were studied in PC12 cells exposed to MCP (10 −5 M) for various time periods. Actin- β was used as loading control to normalize the data. (a) Lane (A): untreated control; (B): Cells exposed to MCP for 6 h; (C): Proteins isolated after 24 h, i.e., 6 h of MCP exposure +18 h without exposure (auto-recovery period); (D): Cells exposed to MCP for 12 h; (E): Cells exposed to MCP for 24 h. (b) Relative quantification of alterations in the expression of different proteins., viz CYP1A1 (59 kDa), CYP1A2 (57 kDa), CYP2B1 (55 kDa), CYP2B2 (54 kDa), CYP2E1 (56 kDa), GSTP1-1 (23.5, 42 and 46 kDa), P 53 (53 kDa), Bax (29 kDa), Bcl 2 (23 kDa), activated caspase-9 (35 kDa), activated caspase-3 (21 kDa), and Actin-β (42 kDa) in PC12 cells exposed to MCP (10 −5 M) for various time periods. Actin-β was used as internal control to normalize the data. Quantification was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase™ FC StandAlone V.4.0 software. * = P

    Journal: PLoS ONE

    Article Title: Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s

    doi: 10.1371/journal.pone.0017757

    Figure Lengend Snippet: Alterations in the expression of proteins involved in the metabolism [figure-6 (i) a b], oxidative stress [figure-6 (ii) a b], and cell death [figure-6 (iii) a b] were studied in PC12 cells exposed to MCP (10 −5 M) for various time periods. Actin- β was used as loading control to normalize the data. (a) Lane (A): untreated control; (B): Cells exposed to MCP for 6 h; (C): Proteins isolated after 24 h, i.e., 6 h of MCP exposure +18 h without exposure (auto-recovery period); (D): Cells exposed to MCP for 12 h; (E): Cells exposed to MCP for 24 h. (b) Relative quantification of alterations in the expression of different proteins., viz CYP1A1 (59 kDa), CYP1A2 (57 kDa), CYP2B1 (55 kDa), CYP2B2 (54 kDa), CYP2E1 (56 kDa), GSTP1-1 (23.5, 42 and 46 kDa), P 53 (53 kDa), Bax (29 kDa), Bcl 2 (23 kDa), activated caspase-9 (35 kDa), activated caspase-3 (21 kDa), and Actin-β (42 kDa) in PC12 cells exposed to MCP (10 −5 M) for various time periods. Actin-β was used as internal control to normalize the data. Quantification was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase™ FC StandAlone V.4.0 software. * = P

    Article Snippet: After blocking (2 h at 37°C), membranes were incubated overnight at 4°C with anti-protein primary antibodies specific for 1A1, 1A2, 2B1/2B2 & 2E1 (1∶500, Chemicon, USA), GSTP1-1 (1∶1000, Calbiochem, USA), p53, Bcl2 , Bax, Activated Caspase-9, Activated Caspase- 3 (1∶1000, CST, USA) and Actin-β (1∶2000, Santa Cruz, USA) in blocking buffer (pH 7.5).

    Techniques: Expressing, Isolation, Software

    Transcriptional changes in the levels of selected xenobiotic metabolizing cytochrome P450s (CYPs) and apoptosis markers in PC12 cells exposed to MCP. (a) MCP-induced alterations in the mRNA expression of marker genes associated with metabolism of xenobiotics in PC12 cells. Quantitative Real Time PCR (RT-PCR q ) was performed in triplicate by TaqMan Probe using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, USA). Actin-β was used as internal control to normalize the data and MCP induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups. (b) MCP induced alterations in the mRNA expression of marker genes associated with apoptosis in PC12 cells. Quantitative Real Time PCR (RT-PCR q ) was performed in triplicate by SYBR Green dye using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, USA). Actin-β was used as internal control to normalize the data and MCP induced alterations in mRNA expression are expressed in relative quantity (RQ) compared with respective unexposed control groups. Reliability of Specific products was checked by melting curve analysis as well as running the product onto 2% agarose Gel.

    Journal: PLoS ONE

    Article Title: Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s

    doi: 10.1371/journal.pone.0017757

    Figure Lengend Snippet: Transcriptional changes in the levels of selected xenobiotic metabolizing cytochrome P450s (CYPs) and apoptosis markers in PC12 cells exposed to MCP. (a) MCP-induced alterations in the mRNA expression of marker genes associated with metabolism of xenobiotics in PC12 cells. Quantitative Real Time PCR (RT-PCR q ) was performed in triplicate by TaqMan Probe using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, USA). Actin-β was used as internal control to normalize the data and MCP induced alterations in mRNA expression are expressed in relative quantity compared with respective unexposed control groups. (b) MCP induced alterations in the mRNA expression of marker genes associated with apoptosis in PC12 cells. Quantitative Real Time PCR (RT-PCR q ) was performed in triplicate by SYBR Green dye using ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, USA). Actin-β was used as internal control to normalize the data and MCP induced alterations in mRNA expression are expressed in relative quantity (RQ) compared with respective unexposed control groups. Reliability of Specific products was checked by melting curve analysis as well as running the product onto 2% agarose Gel.

    Article Snippet: After blocking (2 h at 37°C), membranes were incubated overnight at 4°C with anti-protein primary antibodies specific for 1A1, 1A2, 2B1/2B2 & 2E1 (1∶500, Chemicon, USA), GSTP1-1 (1∶1000, Calbiochem, USA), p53, Bcl2 , Bax, Activated Caspase-9, Activated Caspase- 3 (1∶1000, CST, USA) and Actin-β (1∶2000, Santa Cruz, USA) in blocking buffer (pH 7.5).

    Techniques: Expressing, Marker, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing, SYBR Green Assay, Agarose Gel Electrophoresis

    Effects of MEKS on the activations of apoptosis-related proteins. MDA-MB-231 cells were treated with 0, 25, or 50 μg/ml of MEKS for 4 h or 30 nM paclitaxel for 24 h (positive control). Protein expressions of cleaved caspase 3 (C), 8 (B) and 9 (A), cleaved PARP (D) and beta-actin (the loading control) were detected by Western blotting

    Journal: Pharmacognosy Magazine

    Article Title: Anti-cancer effects of Kochia scoparia fruit in human breast cancer cells

    doi: 10.4103/0973-1296.139812

    Figure Lengend Snippet: Effects of MEKS on the activations of apoptosis-related proteins. MDA-MB-231 cells were treated with 0, 25, or 50 μg/ml of MEKS for 4 h or 30 nM paclitaxel for 24 h (positive control). Protein expressions of cleaved caspase 3 (C), 8 (B) and 9 (A), cleaved PARP (D) and beta-actin (the loading control) were detected by Western blotting

    Article Snippet: The antibodies targeting cleaved caspase 3, cleaved caspase 8, cleaved caspase 9 and cleaved Poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA, USA), while anti-beta actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Multiple Displacement Amplification, Positive Control, Western Blot

    Immunoblotting analysis of N-cadherin, peroxisome proliferator-activated receptor gamma (PPARγ) and beta-actin in control and VD3-treated HN9.10e cells. (A) The position of the 97 kDa for N-cadherin, 57 kDa for PPARG and 43 kDa for beta-actin was evaluated in relation to the position of molecular size standards. (B) The area density was quantified by densitometry scanning and analysis with Scion Image. Data are expressed as percentage variation of VD3-treated HN9.10e cells compared with control HN9.10e cells and represent the mean ± SD of three independent experiments. ∗ P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Effect of Vitamin D in HN9.10e Embryonic Hippocampal Cells and in Hippocampus from MPTP-Induced Parkinson’s Disease Mouse Model

    doi: 10.3389/fncel.2018.00031

    Figure Lengend Snippet: Immunoblotting analysis of N-cadherin, peroxisome proliferator-activated receptor gamma (PPARγ) and beta-actin in control and VD3-treated HN9.10e cells. (A) The position of the 97 kDa for N-cadherin, 57 kDa for PPARG and 43 kDa for beta-actin was evaluated in relation to the position of molecular size standards. (B) The area density was quantified by densitometry scanning and analysis with Scion Image. Data are expressed as percentage variation of VD3-treated HN9.10e cells compared with control HN9.10e cells and represent the mean ± SD of three independent experiments. ∗ P

    Article Snippet: Reagents Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), dithiothreitol, phenylmethylsulfonylfluoride (PMSF) were obtained from Sigma Chemical, Co. (St. Louis, MO, United States); VD3 was obtained from DBA Italia (Segrate, Milan, Italy); anti-GFAP antibody was obtained from Dako, Agilent (Santa Clara, CA, United States), anti-N-cadherin, anti- peroxisome proliferator-activated receptor gamma (PPARγ), anti-VDR from Elabscience (Houston, TX, United States) and anti-beta actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States); anti-neuron specific enolase (NSE) and anti-NF200 antibodies were from NOVOCASTRA Laboratories, Ltd. (Newcastle, United Kingdom).

    Techniques:

    Role of Vif polyubiquitylation in A3G degradation. A , polyubiquitylation of HIV-1 Vif protein in vivo . Wild-type or Lys-free (Vif16K/R) HIV-1 Vif proteins were coexpressed with FLAG-tagged Ub 48A . Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum. B , expression of Lys-free Vif. Equal amounts of expression vectors for wild-type ( WT ) or Lys-free Vif were transfected into 293T cells, and protein expression was determined by Western blotting. C , activity of Lys-free Vif. Wild-type or Lys-free Vif proteins were coexpressed with A3G protein fused with FLuc in 293T cells, and the cellular luciferase activity was determined as before. Error bars represent S.D. in at least three independent experiments. D , interaction of HIV-1 Vif with A3G and Cul5 E3 ligase. Vif proteins were coexpressed with FLAG-tagged A3G or HA-tagged Cul5 or EloC. To detect the interaction between Vif and A3G, proteins were pulled down by anti-FLAG beads and analyzed by Western blotting using anti-FLAG and anti-Vif antibodies. To detect the interaction between Vif and Cul5 or EloC, proteins were immunoprecipitated ( IP ) by anti-Vif polyclonal antibody and analyzed by Western blotting using anti-Vif and anti-HA antibodies. I , input; P , pull down. E , A3G polyubiquitylation by Lys-free Vif. Wild-type A3G was coexpressed with wild-type or Lys-free Vif in the presence of the FLAG-tagged Ub 48A expression vector. Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum.

    Journal: The Journal of Biological Chemistry

    Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *

    doi: 10.1074/jbc.M708728200

    Figure Lengend Snippet: Role of Vif polyubiquitylation in A3G degradation. A , polyubiquitylation of HIV-1 Vif protein in vivo . Wild-type or Lys-free (Vif16K/R) HIV-1 Vif proteins were coexpressed with FLAG-tagged Ub 48A . Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum. B , expression of Lys-free Vif. Equal amounts of expression vectors for wild-type ( WT ) or Lys-free Vif were transfected into 293T cells, and protein expression was determined by Western blotting. C , activity of Lys-free Vif. Wild-type or Lys-free Vif proteins were coexpressed with A3G protein fused with FLuc in 293T cells, and the cellular luciferase activity was determined as before. Error bars represent S.D. in at least three independent experiments. D , interaction of HIV-1 Vif with A3G and Cul5 E3 ligase. Vif proteins were coexpressed with FLAG-tagged A3G or HA-tagged Cul5 or EloC. To detect the interaction between Vif and A3G, proteins were pulled down by anti-FLAG beads and analyzed by Western blotting using anti-FLAG and anti-Vif antibodies. To detect the interaction between Vif and Cul5 or EloC, proteins were immunoprecipitated ( IP ) by anti-Vif polyclonal antibody and analyzed by Western blotting using anti-Vif and anti-HA antibodies. I , input; P , pull down. E , A3G polyubiquitylation by Lys-free Vif. Wild-type A3G was coexpressed with wild-type or Lys-free Vif in the presence of the FLAG-tagged Ub 48A expression vector. Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum.

    Article Snippet: Other antibodies used included horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen), horseradish peroxidase-conjugated anti-HA antibody (Roche Applied Science), polyclonal anti-actin antibody (clone C-11; Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Pierce).

    Techniques: In Vivo, Isolation, Western Blot, Expressing, Transfection, Activity Assay, Luciferase, Immunoprecipitation, Plasmid Preparation

    SMURF2 is a positive mediator of the IL-25-response (a and b) HEK293 cells were transfected with the indicated vectors. After 48 hours the cell lysate was prepared followed by co-immunoprecipitation for 24 hours using antibodies against MYC-tag SMURF2 (a) or V5-tag IL-25R (b). WT and Smurf2 –/– KECs were isolated and infected with Ad-V5- Il-17rb (V5-25R). Cells were then stimulated with IL-25 (100 ng/ml) for the indicated timepoints and lysates prepared and subjected to co-immunoprecipitation with antibody against V5. The co-immunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (a-c) Data are representative results of at least 2 independently performed experiments. (d) WT and Smurf2 –/– mice were injected with IL-25 by intra-tracheal route. After 4 days BAL wash was performed and total cellularity (d) and eosinophil in the BAL (e) were enumerated. (f) Lungs were sectioned and stained as indicated. Representative images from treated mice are shown. (g) RNA was prepared from total lung tissue and RT-qPCR performed for Il-25 and Il-13 normalized to β- actin . (h) IL-13 ELISA from BAL fluid of treated mice. Data presented are from a representative experiment with n=4 mice per experimental group, these experiments were repeated 2 times with similar results. Error bars represent mean ± SEM, * indicates p

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

    Article Title: TRAF4-SMURF2-mediated DAZAP2 degradation is critical for IL-25 signaling and allergic airway inflammation

    doi: 10.4049/jimmunol.1402647

    Figure Lengend Snippet: SMURF2 is a positive mediator of the IL-25-response (a and b) HEK293 cells were transfected with the indicated vectors. After 48 hours the cell lysate was prepared followed by co-immunoprecipitation for 24 hours using antibodies against MYC-tag SMURF2 (a) or V5-tag IL-25R (b). WT and Smurf2 –/– KECs were isolated and infected with Ad-V5- Il-17rb (V5-25R). Cells were then stimulated with IL-25 (100 ng/ml) for the indicated timepoints and lysates prepared and subjected to co-immunoprecipitation with antibody against V5. The co-immunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (a-c) Data are representative results of at least 2 independently performed experiments. (d) WT and Smurf2 –/– mice were injected with IL-25 by intra-tracheal route. After 4 days BAL wash was performed and total cellularity (d) and eosinophil in the BAL (e) were enumerated. (f) Lungs were sectioned and stained as indicated. Representative images from treated mice are shown. (g) RNA was prepared from total lung tissue and RT-qPCR performed for Il-25 and Il-13 normalized to β- actin . (h) IL-13 ELISA from BAL fluid of treated mice. Data presented are from a representative experiment with n=4 mice per experimental group, these experiments were repeated 2 times with similar results. Error bars represent mean ± SEM, * indicates p

    Article Snippet: Antibodies for immunoblots used are as follows; rabbit anti-ACT1 was described previously in , goat anti-TRAF4 (N16), mouse anti-Omni tag, rat anti-IL-17RB (TJ5), rabbit anti-Smurf2 (H50), mouse anti-P-ERK1/2 and goat anti-ACTIN were from Santa Cruz Biotech.

    Techniques: Transfection, Immunoprecipitation, Isolation, Infection, SDS Page, Mouse Assay, Injection, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    IL-25R contains tyrosine residues that modulate its function (a) Schematic diagram showing tyrosine residues on the intracellular domain of IL-17RB and previously mapped DAZAP2 binding region, (right) schematic of SH2 domains on DAZAP2. (b) HEK293 cells were transfected with the indicated vectors, cells were pelleted and lysates were prepared and co-immunoprecipitated with antibodies against HA-tag, following this lysates were subjected to SDS-PAGE and immunoblotting. (c) WT KEC were transduced with Ad- IL-17rb (WT) or Ad- IL-17rb with all intracellular tyrosines mutated to phenylalanine (All-Tyr) for 48 hours followed by SDS-PAGE and immunoblotting. (d) WT KEC were transduced with Ad-IL-17RB and treated with IL-25 for the indicated times, cells were pelleted and lysates were prepared and coimmunoprecipitated with antibody against P-Tyr (upper) or V5 (lower), after which lysates were subjected to SDS-PAGE and immunoblotting. (e) HEK293 cells were transfected with the indicated vectors followed by co-immunoprecipitation against V5-tag and immunoblotting. (f) Il17rb –/– KECs were transduced with adenovirus encoding for IL-17rb WT or Y355F mutant. Cells were pelleted and lysates were prepared and coimmunoprecipitated with antibody against P-Tyr and resolved by SDS-PAGE. (g) Il17rb –/– KECs were transduced with adenovirus encoding for IL-17rb WT or the indicated single Y to F mutants, (top) is immunoblot for the transduced receptors (bottom) KEC were treated for the indicated time with IL-25, RNA was isolated and RT-qPCR performed and normalized to β- actin . (h) HEK293 cells were transfected with the indicated plasmids and Co-immunoprecipitated and resolved on SDS-PAGE followed by immunoblotting as indicated. (i) Traf4 –/– KECs were transduced with adenovirus encoding for IL-17rb WT or the indicated single Y to F mutants, cells were treated for the indicated time with IL-25, RNA was isolated and RT-qPCR performed and normalized to β- actin . Representative data of 2 or 3 independently performed experiments are shown, error bars represent. Error bars represent mean ± SEM.

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

    Article Title: TRAF4-SMURF2-mediated DAZAP2 degradation is critical for IL-25 signaling and allergic airway inflammation

    doi: 10.4049/jimmunol.1402647

    Figure Lengend Snippet: IL-25R contains tyrosine residues that modulate its function (a) Schematic diagram showing tyrosine residues on the intracellular domain of IL-17RB and previously mapped DAZAP2 binding region, (right) schematic of SH2 domains on DAZAP2. (b) HEK293 cells were transfected with the indicated vectors, cells were pelleted and lysates were prepared and co-immunoprecipitated with antibodies against HA-tag, following this lysates were subjected to SDS-PAGE and immunoblotting. (c) WT KEC were transduced with Ad- IL-17rb (WT) or Ad- IL-17rb with all intracellular tyrosines mutated to phenylalanine (All-Tyr) for 48 hours followed by SDS-PAGE and immunoblotting. (d) WT KEC were transduced with Ad-IL-17RB and treated with IL-25 for the indicated times, cells were pelleted and lysates were prepared and coimmunoprecipitated with antibody against P-Tyr (upper) or V5 (lower), after which lysates were subjected to SDS-PAGE and immunoblotting. (e) HEK293 cells were transfected with the indicated vectors followed by co-immunoprecipitation against V5-tag and immunoblotting. (f) Il17rb –/– KECs were transduced with adenovirus encoding for IL-17rb WT or Y355F mutant. Cells were pelleted and lysates were prepared and coimmunoprecipitated with antibody against P-Tyr and resolved by SDS-PAGE. (g) Il17rb –/– KECs were transduced with adenovirus encoding for IL-17rb WT or the indicated single Y to F mutants, (top) is immunoblot for the transduced receptors (bottom) KEC were treated for the indicated time with IL-25, RNA was isolated and RT-qPCR performed and normalized to β- actin . (h) HEK293 cells were transfected with the indicated plasmids and Co-immunoprecipitated and resolved on SDS-PAGE followed by immunoblotting as indicated. (i) Traf4 –/– KECs were transduced with adenovirus encoding for IL-17rb WT or the indicated single Y to F mutants, cells were treated for the indicated time with IL-25, RNA was isolated and RT-qPCR performed and normalized to β- actin . Representative data of 2 or 3 independently performed experiments are shown, error bars represent. Error bars represent mean ± SEM.

    Article Snippet: Antibodies for immunoblots used are as follows; rabbit anti-ACT1 was described previously in , goat anti-TRAF4 (N16), mouse anti-Omni tag, rat anti-IL-17RB (TJ5), rabbit anti-Smurf2 (H50), mouse anti-P-ERK1/2 and goat anti-ACTIN were from Santa Cruz Biotech.

    Techniques: Binding Assay, Transfection, Immunoprecipitation, SDS Page, Transduction, Mutagenesis, Isolation, Quantitative RT-PCR

    Cell-intrinsic IL-25 responses are TRAF4-dependent Naïve CD4-positive T-cells were isolated from WT and Traf4 –/– mice and subsequently activated with plate-bound CD3/CD28 and in the indicated polarizing conditions. (a) ELISA for IL-5 and IL-13 performed from supernatants of activated T-cells. (b) RNA was isolated from activated T-cells in Th0+IL-25 conditions and RT-qPCR for the indicated genes normalized to β -actin . (c) Top, Immunoblot from human airway epithelial cell-line (Bet1a) transfected with siRNA against human Traf4 or scrambled (scr) (100 nM each). Bet1a cells transfected with siRNA were treated with hIL-125 (100 ng/ml) for the indicated times, RNA was isolated and RT-qPCR was performed for the indicated genes normalized to GAPDH . (d) Activated T-cells in Th2 polarizing conditions from WT and Traf4 –/– mice were treated with IL-25 (100 ng/ml) for the indicated times, lysates were subjected to SDS-PAGE followed by immunoblotting for the indicated proteins. (e and f) WT and Traf4 –/– or (f) Act1 –/– , KECs were isolated and infected with Ad-V5- Il-17rb (V5-25R). Cells were then stimulated with IL-25 (100 ng/ml) for the indicated timepoints and lysates prepared and subjected to co-immunoprecipitation with antibody against V5. (g and h) HEK293 cells were transfected with the indicated vectors, followed by coimmunoprecipitation for 24 hours using antibodies against tagged IL-25R. Coimmunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (i) Il-17rb –/– KECs were transduced with the indicated vectors followed by IL-25 stimulation, RT-qPCR was performed and normalized to β -actin . All data are representative of at least 3 independently performed experiments. Error bars represent mean ± SEM, * indicates p

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

    Article Title: TRAF4-SMURF2-mediated DAZAP2 degradation is critical for IL-25 signaling and allergic airway inflammation

    doi: 10.4049/jimmunol.1402647

    Figure Lengend Snippet: Cell-intrinsic IL-25 responses are TRAF4-dependent Naïve CD4-positive T-cells were isolated from WT and Traf4 –/– mice and subsequently activated with plate-bound CD3/CD28 and in the indicated polarizing conditions. (a) ELISA for IL-5 and IL-13 performed from supernatants of activated T-cells. (b) RNA was isolated from activated T-cells in Th0+IL-25 conditions and RT-qPCR for the indicated genes normalized to β -actin . (c) Top, Immunoblot from human airway epithelial cell-line (Bet1a) transfected with siRNA against human Traf4 or scrambled (scr) (100 nM each). Bet1a cells transfected with siRNA were treated with hIL-125 (100 ng/ml) for the indicated times, RNA was isolated and RT-qPCR was performed for the indicated genes normalized to GAPDH . (d) Activated T-cells in Th2 polarizing conditions from WT and Traf4 –/– mice were treated with IL-25 (100 ng/ml) for the indicated times, lysates were subjected to SDS-PAGE followed by immunoblotting for the indicated proteins. (e and f) WT and Traf4 –/– or (f) Act1 –/– , KECs were isolated and infected with Ad-V5- Il-17rb (V5-25R). Cells were then stimulated with IL-25 (100 ng/ml) for the indicated timepoints and lysates prepared and subjected to co-immunoprecipitation with antibody against V5. (g and h) HEK293 cells were transfected with the indicated vectors, followed by coimmunoprecipitation for 24 hours using antibodies against tagged IL-25R. Coimmunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (i) Il-17rb –/– KECs were transduced with the indicated vectors followed by IL-25 stimulation, RT-qPCR was performed and normalized to β -actin . All data are representative of at least 3 independently performed experiments. Error bars represent mean ± SEM, * indicates p

    Article Snippet: Antibodies for immunoblots used are as follows; rabbit anti-ACT1 was described previously in , goat anti-TRAF4 (N16), mouse anti-Omni tag, rat anti-IL-17RB (TJ5), rabbit anti-Smurf2 (H50), mouse anti-P-ERK1/2 and goat anti-ACTIN were from Santa Cruz Biotech.

    Techniques: Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Transfection, SDS Page, Infection, Immunoprecipitation, Transduction

    PRDM14 expression in testicular and intracranial germ cell tumors A. Western blot of nuclear (5 μg) and cytoplasmic (5 μg) extracts from embryonal carcinoma cell (ECC) lines, Ntera2 (NT) and GCT27. Blots probed for PRDM14 (64 kD), OCT4 (45 kD), and beta-actin (42 kD). B. Immunofluorescence of ECC line, GCT27, showing staining for PRDM14/OCT4, top panel. Immunofluorescence of paraffin section of a seminoma tumor sample, showing staining for PRDM14/OCT4, bottom panel. Scale bars, 15 μm. C. Immunohistochemistry of paraffin section of tumor sample containing corresponding GCNIS portion of the tumor, staining for PRDM14, and GCNIS markers OCT4 and PLAP. Scale bars, 15 μm. D. Immunohistochemistry of paraffin section of mixed germ cell tumor, corresponding to embryonal carcinoma, staining for PRDM14, SOX2 and SOX17, top panel. Magnified images of cells demarked in yellow box in top panel. Scale bars, 15 μm (top panel). 5 μm (bottom panel). E. Immunohistochemistry staining of intracranial germinoma, yolk sac and teratoma, for PRDM14. Scale bars, 20 μm.

    Journal: Stem cell research

    Article Title: PRDM14 is expressed in germ cell tumors with constitutive overexpression altering human germline differentiation and proliferation

    doi: 10.1016/j.scr.2017.12.016

    Figure Lengend Snippet: PRDM14 expression in testicular and intracranial germ cell tumors A. Western blot of nuclear (5 μg) and cytoplasmic (5 μg) extracts from embryonal carcinoma cell (ECC) lines, Ntera2 (NT) and GCT27. Blots probed for PRDM14 (64 kD), OCT4 (45 kD), and beta-actin (42 kD). B. Immunofluorescence of ECC line, GCT27, showing staining for PRDM14/OCT4, top panel. Immunofluorescence of paraffin section of a seminoma tumor sample, showing staining for PRDM14/OCT4, bottom panel. Scale bars, 15 μm. C. Immunohistochemistry of paraffin section of tumor sample containing corresponding GCNIS portion of the tumor, staining for PRDM14, and GCNIS markers OCT4 and PLAP. Scale bars, 15 μm. D. Immunohistochemistry of paraffin section of mixed germ cell tumor, corresponding to embryonal carcinoma, staining for PRDM14, SOX2 and SOX17, top panel. Magnified images of cells demarked in yellow box in top panel. Scale bars, 15 μm (top panel). 5 μm (bottom panel). E. Immunohistochemistry staining of intracranial germinoma, yolk sac and teratoma, for PRDM14. Scale bars, 20 μm.

    Article Snippet: Primary antibodies (1:1000) were rabbit-anti-PRDM14 (Abcam, ab187881), goat-anti-OCT3/4 (N-19) (Santa Cruz, sc-8628), mouse-anti-ACTIN (Santa Cruz, sc47778) and rabbit anti-H3 (Abcam ab1791).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Paraffin Section, Immunohistochemistry

    Calcarea carbonica induces T cell-mediated apoptosis of primary mammary tumor. (A) T cells isolated from patient’s peripheral circulation were primed with media-/placebo-/calcarea carbonica-treated tumor supernatant for 72 h and then co-cultured with primary mammary tumor for 48 h. In parallel, primary mammary tumor cells was directly exposed to placebo-/calcarea carbonica for 48 h in the absence of T cells. Tumor cell apoptosis was then scored by Annexin-V-PE/7-AAD-positivity and represented graphically. (B) T cells isolated from normal and cancer patient’s peripheral blood were co-cultured with control mammary tissue and tumor mammary tissue explants, respectively for 48 h and percent T cell apoptosis was scored by Annexin-V-PE/7-AAD-positivity. (C) The same experimental set was analysed for percentage of CD4 + and CD8 + T cells flow cytometrically. (D) Lysates of primary breast cancer cells co-cultured with or without placebo-/calcarea carbonica-primed T cells of same patient’s blood were Western blotted for the analysis of p53, Bax, Bcl-2 and active-caspase-3. α-Actin was used as loading control. *p

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit

    doi: 10.1186/1472-6882-13-230

    Figure Lengend Snippet: Calcarea carbonica induces T cell-mediated apoptosis of primary mammary tumor. (A) T cells isolated from patient’s peripheral circulation were primed with media-/placebo-/calcarea carbonica-treated tumor supernatant for 72 h and then co-cultured with primary mammary tumor for 48 h. In parallel, primary mammary tumor cells was directly exposed to placebo-/calcarea carbonica for 48 h in the absence of T cells. Tumor cell apoptosis was then scored by Annexin-V-PE/7-AAD-positivity and represented graphically. (B) T cells isolated from normal and cancer patient’s peripheral blood were co-cultured with control mammary tissue and tumor mammary tissue explants, respectively for 48 h and percent T cell apoptosis was scored by Annexin-V-PE/7-AAD-positivity. (C) The same experimental set was analysed for percentage of CD4 + and CD8 + T cells flow cytometrically. (D) Lysates of primary breast cancer cells co-cultured with or without placebo-/calcarea carbonica-primed T cells of same patient’s blood were Western blotted for the analysis of p53, Bax, Bcl-2 and active-caspase-3. α-Actin was used as loading control. *p

    Article Snippet: In parallel experiment equivalent amount of protein was Western blotted with anti-α-actin antibody (C-2; Santa Cruz) to confirm equal protein leading.

    Techniques: Isolation, Cell Culture, Flow Cytometry, Western Blot

    Calcarea carbonica triggers T cell-mediated tumor killing via p53-Bax-caspase-3 cascade. (A) EAC, MCF-7 and MDA-MB-231 cells were co-cultured with untreated-/placebo-/calcarea carbonica-primed T cells and subjected to Western blot/RT-PCR analysis to determine the expression profile of p53/Bax/Bcl-2 at protein and Bax/Bcl-2 at mRNA levels (left panels). Right panels represent quantitative data for Western blot. (B) Graphical representation of Bcl-2/Bax protein ratio in tumor cells co-cultured with calcarea carbonica-primed T cells. (C) Wild-type p53-expressing cells were transfected with p53-siRNA (inset) and scored for percent apoptosis when co-cultured with calcarea carbonica-primed T cells. (D) Bax and cytochrome c levels were determined in cytosolic and mitochondrial fractions of tumor cells co-cultured with placebo-/calcarea carbonica-primed T cells by Western blot analysis (left panels). Middle panels represent quantitative data. α-Actin and MnSOD were used as internal protein markers (right panels). (E) Graphical representation of mitochondrial trans-membrane potential of tumor cells co-cultured with calcarea carbonica-primed T cells pre-treated with cyc losporine-A. (F) Expression profiles of pro-/active- forms of caspase-3 in EAC and HBL-100 cells and pro-/active caspase-9 in caspase-3-null MCF-7 cells co-cultured with calcarea carbonica-primed T cells. Right panels represent quantitative data. (G) Expression profiles of active caspase-3 in EAC and caspase-9 in MCF-7 cells co-cultured with calcarea carbonica-primed T cells pre-treated with cyclosporine-A (left panel). Middle panel represent quantitative data. In parallel set, cells were scored for percentage apoptosis (right panel). (H) Percent apoptosis of EAC and HBL-100 cells co-cultured with calcarea carbonica-primed T cells in the presence of caspase-3 inhibitor (Z-DEVD-FMK) or transfected with caspase-3-siRNA and percent apoptosis of MCF-7 cells co-cultured with calcarea carbonica-primed T cells in the presence of caspase-9 inhibitor (Z-LEHD-FMK). Values are mean ±SEM of five independent experiments. *p

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit

    doi: 10.1186/1472-6882-13-230

    Figure Lengend Snippet: Calcarea carbonica triggers T cell-mediated tumor killing via p53-Bax-caspase-3 cascade. (A) EAC, MCF-7 and MDA-MB-231 cells were co-cultured with untreated-/placebo-/calcarea carbonica-primed T cells and subjected to Western blot/RT-PCR analysis to determine the expression profile of p53/Bax/Bcl-2 at protein and Bax/Bcl-2 at mRNA levels (left panels). Right panels represent quantitative data for Western blot. (B) Graphical representation of Bcl-2/Bax protein ratio in tumor cells co-cultured with calcarea carbonica-primed T cells. (C) Wild-type p53-expressing cells were transfected with p53-siRNA (inset) and scored for percent apoptosis when co-cultured with calcarea carbonica-primed T cells. (D) Bax and cytochrome c levels were determined in cytosolic and mitochondrial fractions of tumor cells co-cultured with placebo-/calcarea carbonica-primed T cells by Western blot analysis (left panels). Middle panels represent quantitative data. α-Actin and MnSOD were used as internal protein markers (right panels). (E) Graphical representation of mitochondrial trans-membrane potential of tumor cells co-cultured with calcarea carbonica-primed T cells pre-treated with cyc losporine-A. (F) Expression profiles of pro-/active- forms of caspase-3 in EAC and HBL-100 cells and pro-/active caspase-9 in caspase-3-null MCF-7 cells co-cultured with calcarea carbonica-primed T cells. Right panels represent quantitative data. (G) Expression profiles of active caspase-3 in EAC and caspase-9 in MCF-7 cells co-cultured with calcarea carbonica-primed T cells pre-treated with cyclosporine-A (left panel). Middle panel represent quantitative data. In parallel set, cells were scored for percentage apoptosis (right panel). (H) Percent apoptosis of EAC and HBL-100 cells co-cultured with calcarea carbonica-primed T cells in the presence of caspase-3 inhibitor (Z-DEVD-FMK) or transfected with caspase-3-siRNA and percent apoptosis of MCF-7 cells co-cultured with calcarea carbonica-primed T cells in the presence of caspase-9 inhibitor (Z-LEHD-FMK). Values are mean ±SEM of five independent experiments. *p

    Article Snippet: In parallel experiment equivalent amount of protein was Western blotted with anti-α-actin antibody (C-2; Santa Cruz) to confirm equal protein leading.

    Techniques: Multiple Displacement Amplification, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

    Downregulation of BIRC5 (survivin) over time in MNV-1-infected RAW264.7 cells. The levels of BIRC5 and survivin, as well as the level of genomic MNV-1 RNA, are shown. Survivin transcription in MNV-1-infected cells compared to mock-infected cells (24 h p.i.) was normalized to the level of ß-actin transcription. MNV-1 genomic RNA detection by quantitative real-time PCR is plotted in comparison to the decreasing levels of BIRC5. Survivin was detected by Western blot analyses of infected cells corresponding to the same time points of MNV-1 infection. Equivalent protein amounts were loaded in each gel and detected with anti-PCNA antibody.

    Journal: Journal of Virology

    Article Title: Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin ▿

    doi: 10.1128/JVI.02028-08

    Figure Lengend Snippet: Downregulation of BIRC5 (survivin) over time in MNV-1-infected RAW264.7 cells. The levels of BIRC5 and survivin, as well as the level of genomic MNV-1 RNA, are shown. Survivin transcription in MNV-1-infected cells compared to mock-infected cells (24 h p.i.) was normalized to the level of ß-actin transcription. MNV-1 genomic RNA detection by quantitative real-time PCR is plotted in comparison to the decreasing levels of BIRC5. Survivin was detected by Western blot analyses of infected cells corresponding to the same time points of MNV-1 infection. Equivalent protein amounts were loaded in each gel and detected with anti-PCNA antibody.

    Article Snippet: Anti-PCNA and anti-ß-actin antibodies were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, and Sigma-Aldrich, St. Louis, MO, respectively.

    Techniques: Infection, RNA Detection, Real-time Polymerase Chain Reaction, Western Blot

    MNV-1 infection triggers the mitochondrial pathway of apoptosis. Cytosolic and mitochondrion-enriched protein fractions were isolated from mock- and MNV-1-infected RAW264.7 cells collected at 16 h p.i. and at 8 and 16 h p.i., respectively. (A) The collected cytosolic proteins were probed with antibodies specific to mitochondrial marker protein COX IV to verify the absence of contamination with mitochondrial proteins. (B) The loading amounts of mitochondrial and cytosolic protein samples were normalized using total protein content and verified by probing with ß-actin-specific antibodies. (C) Cytochrome c release from the mitochondria was examined using normalized amounts of cytosolic (lanes 1, 3, and 5) and mitochondrial (lanes 2, 4, and 6) protein samples and cytochrome c -specific antibodies. Quantification of the cytochrome c release was performed using densitometry with the help of ImageQuant software (Molecular Dynamics).

    Journal: Journal of Virology

    Article Title: Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin ▿

    doi: 10.1128/JVI.02028-08

    Figure Lengend Snippet: MNV-1 infection triggers the mitochondrial pathway of apoptosis. Cytosolic and mitochondrion-enriched protein fractions were isolated from mock- and MNV-1-infected RAW264.7 cells collected at 16 h p.i. and at 8 and 16 h p.i., respectively. (A) The collected cytosolic proteins were probed with antibodies specific to mitochondrial marker protein COX IV to verify the absence of contamination with mitochondrial proteins. (B) The loading amounts of mitochondrial and cytosolic protein samples were normalized using total protein content and verified by probing with ß-actin-specific antibodies. (C) Cytochrome c release from the mitochondria was examined using normalized amounts of cytosolic (lanes 1, 3, and 5) and mitochondrial (lanes 2, 4, and 6) protein samples and cytochrome c -specific antibodies. Quantification of the cytochrome c release was performed using densitometry with the help of ImageQuant software (Molecular Dynamics).

    Article Snippet: Anti-PCNA and anti-ß-actin antibodies were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, and Sigma-Aldrich, St. Louis, MO, respectively.

    Techniques: Infection, Isolation, Marker, Software

    IFN-α down-regulates telomerase activity in the cytoplasm and the nucleus of CD8 + T cells. A representative gel showing telomerase activity (TRAP Assay, see Material and Methods) of cytoplasmic and nuclear extracts from 5 × 10 3 viable CD8 + T cells tested 72 hours following activation with anti CD3 Ab plus rhIL-2 is shown in A. Graph showing pooled results of the effect of IFN-α on telomerase activity of fractioned extracts from 3 separate donors is shown in B. A representative experiment showing the effect of IFN-α on hTERT expression, tested on cytoplasmic and nuclear extracts from 5×10 6 viable CD8 + T lymphocytes 36 hours following activation by Western Blot is shown in C. Quality of extracts was tested using anti β actin and Histone H1 Ab respectively. The graph in the panel D shows the mean ± SD of the ratio protein/β-actin and Histone H1 for cytoplasmic and nuclear compartments respectively, for 3 donors. The effect of IFN-α on the distribution and composition of NPCs was tested on CD8 + ). In panel E a representative image of the staining is shown. The images were collected on an inverted confocal microscope (Olympus) with a 60X objective (N.A. 1.45) with a SIM scanner using the Fluoview software. The fluorescence within a region of interest surrounding the nuclear envelope in twelve mid-plane sections was measured. All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

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

    Article Title: IFN-? Inhibits Telomerase in Human CD8+ T Cells by Both hTERT Downregulation and Induction of p38 MAPkinase Signalling

    doi: 10.4049/jimmunol.1301409

    Figure Lengend Snippet: IFN-α down-regulates telomerase activity in the cytoplasm and the nucleus of CD8 + T cells. A representative gel showing telomerase activity (TRAP Assay, see Material and Methods) of cytoplasmic and nuclear extracts from 5 × 10 3 viable CD8 + T cells tested 72 hours following activation with anti CD3 Ab plus rhIL-2 is shown in A. Graph showing pooled results of the effect of IFN-α on telomerase activity of fractioned extracts from 3 separate donors is shown in B. A representative experiment showing the effect of IFN-α on hTERT expression, tested on cytoplasmic and nuclear extracts from 5×10 6 viable CD8 + T lymphocytes 36 hours following activation by Western Blot is shown in C. Quality of extracts was tested using anti β actin and Histone H1 Ab respectively. The graph in the panel D shows the mean ± SD of the ratio protein/β-actin and Histone H1 for cytoplasmic and nuclear compartments respectively, for 3 donors. The effect of IFN-α on the distribution and composition of NPCs was tested on CD8 + ). In panel E a representative image of the staining is shown. The images were collected on an inverted confocal microscope (Olympus) with a 60X objective (N.A. 1.45) with a SIM scanner using the Fluoview software. The fluorescence within a region of interest surrounding the nuclear envelope in twelve mid-plane sections was measured. All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

    Article Snippet: All filters were probed with anti β actin Ab (Santa Cruz) as loading control.

    Techniques: Activity Assay, TRAP Assay, Activation Assay, Expressing, Western Blot, Staining, Microscopy, Software, Fluorescence

    IFN-α activates MAPK p38 signalling. A representative experiment showing the effect of IFN-α on p-38 Thr180/182 MAPK and total p38 MAPK expression tested on whole cell extracts of 2×10 6 viable CD8 + T lymphocytes 24 hours following activation with anti CD3 Ab plus rhIL-2 by Western Blot is shown in A. Gel loading control was performed analyzing β actin expression. IFN-α treatment induces activation of p38 through phosphorylation of Thr180/Tyr182. Panel B shows the mean of the ratio ± SD of p-38 Thr180/182 to total p38 for 3 donors. The role of p38 in IFN-α mediated inhibition of telomerase activity is evaluated by a p38 inhibition experiment by blocking p38 signalling in stimulated T cells by the addition of BIRB796 (BIRB), an inhibitor that blocks the activation of all four of the isoforms of p38. Telomerase activity measured by ELISA is shown as proportional to the OD at 450 nm after due normalization against a negative control (heat-inactivated telomerase sample). p38 blockade enhances cell telomerase activity in CD8 + T cells (C). All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

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

    Article Title: IFN-? Inhibits Telomerase in Human CD8+ T Cells by Both hTERT Downregulation and Induction of p38 MAPkinase Signalling

    doi: 10.4049/jimmunol.1301409

    Figure Lengend Snippet: IFN-α activates MAPK p38 signalling. A representative experiment showing the effect of IFN-α on p-38 Thr180/182 MAPK and total p38 MAPK expression tested on whole cell extracts of 2×10 6 viable CD8 + T lymphocytes 24 hours following activation with anti CD3 Ab plus rhIL-2 by Western Blot is shown in A. Gel loading control was performed analyzing β actin expression. IFN-α treatment induces activation of p38 through phosphorylation of Thr180/Tyr182. Panel B shows the mean of the ratio ± SD of p-38 Thr180/182 to total p38 for 3 donors. The role of p38 in IFN-α mediated inhibition of telomerase activity is evaluated by a p38 inhibition experiment by blocking p38 signalling in stimulated T cells by the addition of BIRB796 (BIRB), an inhibitor that blocks the activation of all four of the isoforms of p38. Telomerase activity measured by ELISA is shown as proportional to the OD at 450 nm after due normalization against a negative control (heat-inactivated telomerase sample). p38 blockade enhances cell telomerase activity in CD8 + T cells (C). All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

    Article Snippet: All filters were probed with anti β actin Ab (Santa Cruz) as loading control.

    Techniques: Expressing, Activation Assay, Western Blot, Inhibition, Activity Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    IFN-α mediates down regulation of AKT signalling. A representative experiment showing the effect of IFN-α on, p-Ser 473 -AKT, p-Thr 308 -AKT, total AKT, p-Tyr 307 -PP2A, total PP2A expression tested on whole cell extracts of 2×10 6 viable CD8 + T lymphocytes 24 hours following activation with anti CD3 Ab plus rhIL-2 by Western Blot is shown in A. IFN-α reduces phosphorylation of AKT on both Ser 473 and Thr 308 sites, while total AKT is not affected. IFN-α treatment up-regulates total PP2A and decreases the inactive form Tyr 307 -PP2A. Gel loading control was performed analyzing β actin expression. Graphs of the mean ± SD of the ratio of band intensity values of Ser 473 -AKT and Thr 30 -AKT to total AKT and pTyr 307- PP2A to total PP2A respectively for 3 donors are shown in B. All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

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

    Article Title: IFN-? Inhibits Telomerase in Human CD8+ T Cells by Both hTERT Downregulation and Induction of p38 MAPkinase Signalling

    doi: 10.4049/jimmunol.1301409

    Figure Lengend Snippet: IFN-α mediates down regulation of AKT signalling. A representative experiment showing the effect of IFN-α on, p-Ser 473 -AKT, p-Thr 308 -AKT, total AKT, p-Tyr 307 -PP2A, total PP2A expression tested on whole cell extracts of 2×10 6 viable CD8 + T lymphocytes 24 hours following activation with anti CD3 Ab plus rhIL-2 by Western Blot is shown in A. IFN-α reduces phosphorylation of AKT on both Ser 473 and Thr 308 sites, while total AKT is not affected. IFN-α treatment up-regulates total PP2A and decreases the inactive form Tyr 307 -PP2A. Gel loading control was performed analyzing β actin expression. Graphs of the mean ± SD of the ratio of band intensity values of Ser 473 -AKT and Thr 30 -AKT to total AKT and pTyr 307- PP2A to total PP2A respectively for 3 donors are shown in B. All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

    Article Snippet: All filters were probed with anti β actin Ab (Santa Cruz) as loading control.

    Techniques: Expressing, Activation Assay, Western Blot

    IFN-α down-regulates telomerase activity in CD8 + T cells. A representative blot of telomerase activity (TRAP Assay, see Material and Methods) of whole cell extracts from 5 × 10 3 viable CD8 + T cells determined 72 hours following activation with anti CD3 Ab plus rhIL-2 is shown in A. Graph showing pooled results of the effect of IFN-α on telomerase activity from 3 separate donors is shown in B. The effect of IFN-α (500 IU/ml) on hTERT mRNA expression of CD8 + T lymphocytes 24 hours following activation with anti CD3 Ab plus rhIL-2 analyzed by quantitative real-time RT-PCR is shown in C. Levels of hTERT are normalized against ACTB housekeeping gene expression. The graph shows the difference in terms of gene expression working out the Delta Delta CT algorithm between TERT and the housekeeping ACTB. Data shown are representative of 3 independent experiments. The effect of IFN-α on hTERT expression tested on whole cell extracts of 2×10 6 viable CD8 + T lymphocytes 36 hours following activation with or without anti CD3 Ab plus rhIL-2 was analyzed by Western Blot. A representative Blot is shown in D. IFN-α reduces total cell expression of hTERT in CD8 + T cells. Gel loading control was based on β actin expression. Graph showing the mean ± SD of the ratio protein/β-actin band intensity values for 3 donors is shown in E. All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

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

    Article Title: IFN-? Inhibits Telomerase in Human CD8+ T Cells by Both hTERT Downregulation and Induction of p38 MAPkinase Signalling

    doi: 10.4049/jimmunol.1301409

    Figure Lengend Snippet: IFN-α down-regulates telomerase activity in CD8 + T cells. A representative blot of telomerase activity (TRAP Assay, see Material and Methods) of whole cell extracts from 5 × 10 3 viable CD8 + T cells determined 72 hours following activation with anti CD3 Ab plus rhIL-2 is shown in A. Graph showing pooled results of the effect of IFN-α on telomerase activity from 3 separate donors is shown in B. The effect of IFN-α (500 IU/ml) on hTERT mRNA expression of CD8 + T lymphocytes 24 hours following activation with anti CD3 Ab plus rhIL-2 analyzed by quantitative real-time RT-PCR is shown in C. Levels of hTERT are normalized against ACTB housekeeping gene expression. The graph shows the difference in terms of gene expression working out the Delta Delta CT algorithm between TERT and the housekeeping ACTB. Data shown are representative of 3 independent experiments. The effect of IFN-α on hTERT expression tested on whole cell extracts of 2×10 6 viable CD8 + T lymphocytes 36 hours following activation with or without anti CD3 Ab plus rhIL-2 was analyzed by Western Blot. A representative Blot is shown in D. IFN-α reduces total cell expression of hTERT in CD8 + T cells. Gel loading control was based on β actin expression. Graph showing the mean ± SD of the ratio protein/β-actin band intensity values for 3 donors is shown in E. All p values were calculated using one-way paired Student’s t -test. Asterisk indicates p

    Article Snippet: All filters were probed with anti β actin Ab (Santa Cruz) as loading control.

    Techniques: Activity Assay, TRAP Assay, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

    SAM inhibits the LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with SAM (0.5 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. β-Actin was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.

    Journal: ACS Omega

    Article Title: Methionine Attenuates Lipopolysaccharide-Induced Inflammatory Responses via DNA Methylation in Macrophages

    doi: 10.1021/acsomega.8b03571

    Figure Lengend Snippet: SAM inhibits the LPS-induced phosphorylation of MAPKs in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with SAM (0.5 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. β-Actin was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.

    Article Snippet: An Ab against β-actin was purchased from Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Cell Culture, Western Blot

    Met inhibits the LPS-induced phosphorylation of mitogen-activated protein kinases (MAPK) in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with Met (10 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. β-Actin was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.

    Journal: ACS Omega

    Article Title: Methionine Attenuates Lipopolysaccharide-Induced Inflammatory Responses via DNA Methylation in Macrophages

    doi: 10.1021/acsomega.8b03571

    Figure Lengend Snippet: Met inhibits the LPS-induced phosphorylation of mitogen-activated protein kinases (MAPK) in RAW 264.7 macrophages. RAW 264.7 cells were cultured for 12 h with Met (10 mM) and then treated with LPS (100 ng/mL) for 0, 5, 15, 30, and 60 min. Western blotting assays for phospho-ERK1/2 (p-ERK1/2), ERK1/2, p-JNK, JNK, p-p38, and p38 were performed. β-Actin was used as the loading control. Levels for target phosphorylated proteins were normalized to total proteins. All data shown are representative of three independent experiments.

    Article Snippet: An Ab against β-actin was purchased from Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Cell Culture, Western Blot

    Detection of PMCA4a in reproductive luminal fluids and its acquisition on caudal sperm A ) Representative Western blot of FLFs collected during pro-estrus and estrus and metestrus and diestrus (40 µg proteins loaded). The ~128 kDa PMCA4a is seen in pro-estrus and estrus and is marginally present at metestrus and diestrus. Caudal epididymal luminal fluid was used as a positive control. The membrane was stripped and re-probed for HSC70 as a loading control. B ) Western blots of VLF, ULF, and OLF recovered after superovulation demonstrate the presence of the ~128 kDa PMCA4a. Sperm protein was used as a positive control. The membrane was stripped and re-probed for β-actin as a loading control. C ) Quantitation of Western blot data shown in B; the relative expression was determined using VLF as 1. The data represent the mean (±SEM) of a minimum of three independent experiments, and the intensity was quantified by Image J software. ANOVA and t -tests were performed on the mean and P values were calculated. * P = 0.03 indicates a significantly increase amount of PMCA4a in OLF compared to that in VLF. D ) A peak shift of fluorescence intensity to the right, indicates increase amounts of PMCA4a in sperm incubated in FLF compared to PBS for 2 h and treated as described in Materials and Methods.

    Journal: PLoS ONE

    Article Title: Expression and Secretion of Plasma Membrane Ca2+-ATPase 4a (PMCA4a) during Murine Estrus: Association with Oviductal Exosomes and Uptake in Sperm

    doi: 10.1371/journal.pone.0080181

    Figure Lengend Snippet: Detection of PMCA4a in reproductive luminal fluids and its acquisition on caudal sperm A ) Representative Western blot of FLFs collected during pro-estrus and estrus and metestrus and diestrus (40 µg proteins loaded). The ~128 kDa PMCA4a is seen in pro-estrus and estrus and is marginally present at metestrus and diestrus. Caudal epididymal luminal fluid was used as a positive control. The membrane was stripped and re-probed for HSC70 as a loading control. B ) Western blots of VLF, ULF, and OLF recovered after superovulation demonstrate the presence of the ~128 kDa PMCA4a. Sperm protein was used as a positive control. The membrane was stripped and re-probed for β-actin as a loading control. C ) Quantitation of Western blot data shown in B; the relative expression was determined using VLF as 1. The data represent the mean (±SEM) of a minimum of three independent experiments, and the intensity was quantified by Image J software. ANOVA and t -tests were performed on the mean and P values were calculated. * P = 0.03 indicates a significantly increase amount of PMCA4a in OLF compared to that in VLF. D ) A peak shift of fluorescence intensity to the right, indicates increase amounts of PMCA4a in sperm incubated in FLF compared to PBS for 2 h and treated as described in Materials and Methods.

    Article Snippet: Anti-CD9 antibody (SC-18869) and anti-human HSC70 (heat shock cognate protein 70; SC7298) mouse monoclonal antibody were obtained from Santa Cruz Biotechnology, and β-actin antibody (#4970) from Cell Signaling (Boston, MA).

    Techniques: Western Blot, Positive Control, Quantitation Assay, Expressing, Software, Fluorescence, Incubation