anti gapdh  (Abcam)

 
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    Anti GAPDH antibody 6C5
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    ab8245
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    Structured Review

    Abcam anti gapdh
    QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of <t>SMAD2</t> in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; <t>GAPDH,</t> glyceraldehyde 3-phosphate dehydrogenase.

    https://www.bioz.com/result/anti gapdh/product/Abcam
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    Images

    1) Product Images from "circSMAD2 inhibits the epithelial–mesenchymal transition by targeting miR-629 in hepatocellular carcinoma"

    Article Title: circSMAD2 inhibits the epithelial–mesenchymal transition by targeting miR-629 in hepatocellular carcinoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S158008

    QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
    Figure Legend Snippet: QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Techniques Used: Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Negative Control

    2) Product Images from "SENP2 exerts an anti-tumor effect on chronic lymphocytic leukemia cells through the inhibition of the Notch and NF-κB signaling pathways"

    Article Title: SENP2 exerts an anti-tumor effect on chronic lymphocytic leukemia cells through the inhibition of the Notch and NF-κB signaling pathways

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2018.4635

    SENP2 suppresses the Notch and NF-κB signaling pathways. (A) SENP2-overexpressingMEC2 cells and the control cells were cultured in 6 cm dishes and they were harvested after reaching 80-90% confluence, and these cells were then examind by western blot analysis to detect the expression levels of IKKβ, IKKα, IκBα, Cyld, C-myc, β-catenin, Notch1, p50, p65 and SENP2. (B) MEC2 cells in which SENP2 was silenced and the control cells were treated as described in (A). GAPDH was used to confirm equal amount of proteins loaded in each lane. NC-OE-SENP2, MEC2 cells transfected with null (control) overexpression vector; OE-SENP2, MEC2 cells transfected with SENP2 overexpression vector; NC-shRNA-SENP2, MEC2 cells transfected with null control shRNA; shRNA-SENP2, MEC2 cells transfected with shRNA against SENP2.
    Figure Legend Snippet: SENP2 suppresses the Notch and NF-κB signaling pathways. (A) SENP2-overexpressingMEC2 cells and the control cells were cultured in 6 cm dishes and they were harvested after reaching 80-90% confluence, and these cells were then examind by western blot analysis to detect the expression levels of IKKβ, IKKα, IκBα, Cyld, C-myc, β-catenin, Notch1, p50, p65 and SENP2. (B) MEC2 cells in which SENP2 was silenced and the control cells were treated as described in (A). GAPDH was used to confirm equal amount of proteins loaded in each lane. NC-OE-SENP2, MEC2 cells transfected with null (control) overexpression vector; OE-SENP2, MEC2 cells transfected with SENP2 overexpression vector; NC-shRNA-SENP2, MEC2 cells transfected with null control shRNA; shRNA-SENP2, MEC2 cells transfected with shRNA against SENP2.

    Techniques Used: Cell Culture, Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, shRNA

    3) Product Images from "Inhibition of Ep3 attenuates migration and promotes apoptosis of non-small cell lung cancer cells via suppression of TGF-β/Smad signaling"

    Article Title: Inhibition of Ep3 attenuates migration and promotes apoptosis of non-small cell lung cancer cells via suppression of TGF-β/Smad signaling

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.9391

    Inhibition of Ep3 suppresses the growth of A549 cells via inhibition of TGF-β/Smad signaling. The protein expression of (A) TGF-β, (B) p-Smad2 and Smad2, and (C) p-Smad3 and Smad3 were detected by western blotting and normalized to GAPDH levels. (D) Cell viability was assessed by MTT assay. (E) Cell apoptosis was assessed by flow cytometry assay. The data are presented as the mean ± the standard error of the mean of three independent experiments *P
    Figure Legend Snippet: Inhibition of Ep3 suppresses the growth of A549 cells via inhibition of TGF-β/Smad signaling. The protein expression of (A) TGF-β, (B) p-Smad2 and Smad2, and (C) p-Smad3 and Smad3 were detected by western blotting and normalized to GAPDH levels. (D) Cell viability was assessed by MTT assay. (E) Cell apoptosis was assessed by flow cytometry assay. The data are presented as the mean ± the standard error of the mean of three independent experiments *P

    Techniques Used: Inhibition, Expressing, Western Blot, MTT Assay, Flow Cytometry, Cytometry

    4) Product Images from "The combination of NVP-BEZ235 and rapamycin regulates nasopharyngeal carcinoma cell viability and apoptosis via the PI3K/AKT/mTOR pathway"

    Article Title: The combination of NVP-BEZ235 and rapamycin regulates nasopharyngeal carcinoma cell viability and apoptosis via the PI3K/AKT/mTOR pathway

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2018.6896

    NVP-BEZ235 and rapamycin repressed the PI3K/AKT/mTOR pathway. SUNE1 cells were treated with 100 nM NVP-BEZ235, 100 nM rapamycin, or 100 nM NVP-BEZ235 and 100 nM rapamycin for 48 h. The control group was administered the same amount of vehicle. Total RNA from the four groups was isolated for reverse transcription-quantitative polymerase chain reaction. The mRNA levels of (A) PI3K, (B) AKT and (C) mTOR were normalized to the internal control GAPDH. (D) The protein levels of PI3K, AKT, p-AKT, mTOR and p-mTOR were detected by western blotting. (E-K) The protein density of PI3K, AKT, p-AKT, mTOR and p-mTOR was calculated relative to the internal control GAPDH, and the p-AKT/AKT and p-mTOR/mTOR ratios were calculated. **P
    Figure Legend Snippet: NVP-BEZ235 and rapamycin repressed the PI3K/AKT/mTOR pathway. SUNE1 cells were treated with 100 nM NVP-BEZ235, 100 nM rapamycin, or 100 nM NVP-BEZ235 and 100 nM rapamycin for 48 h. The control group was administered the same amount of vehicle. Total RNA from the four groups was isolated for reverse transcription-quantitative polymerase chain reaction. The mRNA levels of (A) PI3K, (B) AKT and (C) mTOR were normalized to the internal control GAPDH. (D) The protein levels of PI3K, AKT, p-AKT, mTOR and p-mTOR were detected by western blotting. (E-K) The protein density of PI3K, AKT, p-AKT, mTOR and p-mTOR was calculated relative to the internal control GAPDH, and the p-AKT/AKT and p-mTOR/mTOR ratios were calculated. **P

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

    5) Product Images from "Endogenous Retrotransposition Activates Oncogenic Pathways in Hepatocellular Carcinoma"

    Article Title: Endogenous Retrotransposition Activates Oncogenic Pathways in Hepatocellular Carcinoma

    Journal: Cell

    doi: 10.1016/j.cell.2013.02.032

    Downregulation of MCC (A) Relative expression of MCC and CTNNB1 in control liver tissue compared to tumor and nontumor liver tissue from donors 33, 70, 95, and 116. An immunoblot performed against anti-MCC, anti-CTNNB1, and anti-GAPDH (loading control) antibodies detected strong MCC expression only in controls and strong CTNNB1 expression only in MCC mutant donors. MCC was also detected weakly in donor 70NT, in line with qRT-PCR results shown in (B). Expected protein molecular weights are marked on right. Note: anti-MCC and anti-CTNNB1 antibodies produce double bands. See Figure S1 for donor 116 CTNNB1 immunohistochemistry. (B) Downregulation of MCC transcription in MCC mutant donors: qRT-PCR revealed that MCC mRNA was significantly reduced compared to control liver tissue in donors 33 (tumor only), 70 (tumor only), 95 (tumor and nontumor), and 116 (tumor and nontumor). ∗∗ p
    Figure Legend Snippet: Downregulation of MCC (A) Relative expression of MCC and CTNNB1 in control liver tissue compared to tumor and nontumor liver tissue from donors 33, 70, 95, and 116. An immunoblot performed against anti-MCC, anti-CTNNB1, and anti-GAPDH (loading control) antibodies detected strong MCC expression only in controls and strong CTNNB1 expression only in MCC mutant donors. MCC was also detected weakly in donor 70NT, in line with qRT-PCR results shown in (B). Expected protein molecular weights are marked on right. Note: anti-MCC and anti-CTNNB1 antibodies produce double bands. See Figure S1 for donor 116 CTNNB1 immunohistochemistry. (B) Downregulation of MCC transcription in MCC mutant donors: qRT-PCR revealed that MCC mRNA was significantly reduced compared to control liver tissue in donors 33 (tumor only), 70 (tumor only), 95 (tumor and nontumor), and 116 (tumor and nontumor). ∗∗ p

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Immunohistochemistry

    6) Product Images from "CAPS1 Negatively Regulates Hepatocellular Carcinoma Development through Alteration of Exocytosis-Associated Tumor Microenvironment"

    Article Title: CAPS1 Negatively Regulates Hepatocellular Carcinoma Development through Alteration of Exocytosis-Associated Tumor Microenvironment

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17101626

    CAPS1 overexpression changed microenvironment in HCC cells. ( A ) The levels of stromal cell-derived factor 1 (SDF-1), hepatocyte growth factor (HGF), and transforming growth factor-beta (TGF-β) were determined by enzyme-linked immunosorbent assay (ELISA) in culture supernatant derived from Huh7 cells stably transfected with control vector or CAPS1; ( B ) Huh7 cells were treated with the culture supernatant from Huh7 cells stably transfected with control vector or CAPS1. Forty-eight hours later, the expression of proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation, was detected by Western blotting. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. * p
    Figure Legend Snippet: CAPS1 overexpression changed microenvironment in HCC cells. ( A ) The levels of stromal cell-derived factor 1 (SDF-1), hepatocyte growth factor (HGF), and transforming growth factor-beta (TGF-β) were determined by enzyme-linked immunosorbent assay (ELISA) in culture supernatant derived from Huh7 cells stably transfected with control vector or CAPS1; ( B ) Huh7 cells were treated with the culture supernatant from Huh7 cells stably transfected with control vector or CAPS1. Forty-eight hours later, the expression of proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation, was detected by Western blotting. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. * p

    Techniques Used: Over Expression, Derivative Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Marker, Western Blot

    7) Product Images from "A Modified Chinese Herbal Decoction (Kai-Xin-San) Promotes NGF-Induced Neuronal Differentiation in PC12 Cells via Up-Regulating Trk A Signaling"

    Article Title: A Modified Chinese Herbal Decoction (Kai-Xin-San) Promotes NGF-Induced Neuronal Differentiation in PC12 Cells via Up-Regulating Trk A Signaling

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2017.00118

    K252a inhibits KXS 2012 -promoted neurofilament expression. (A) K252a (0.1 μM) was applied onto cultured PC12 cells, 3 h before KXS 2012 (15 μg/mL) treatment. After 48 h, PC12 cells were washed by PBS twice and treated with NGF (1.5 ng/mL) for 24 h. (B) K252a (0.1 μM) was applied onto cultured PC12 cells 3 h before KXS 2012 (15 μg/mL) treatment together with NGF (1.5 ng/mL) for 24 h. The cell lysates were collected to determine the expression of NF68. GAPDH served as a loading control. Quantification plot was shown in low panel. Values are expressed as × Basal, where control value is set as 1, Mean ± SEM, n = 4. * p
    Figure Legend Snippet: K252a inhibits KXS 2012 -promoted neurofilament expression. (A) K252a (0.1 μM) was applied onto cultured PC12 cells, 3 h before KXS 2012 (15 μg/mL) treatment. After 48 h, PC12 cells were washed by PBS twice and treated with NGF (1.5 ng/mL) for 24 h. (B) K252a (0.1 μM) was applied onto cultured PC12 cells 3 h before KXS 2012 (15 μg/mL) treatment together with NGF (1.5 ng/mL) for 24 h. The cell lysates were collected to determine the expression of NF68. GAPDH served as a loading control. Quantification plot was shown in low panel. Values are expressed as × Basal, where control value is set as 1, Mean ± SEM, n = 4. * p

    Techniques Used: Expressing, Cell Culture

    KXS 2012 does not induce neuronal differentiation. (A) KXS 2012 (5–25 μg/mL) were applied onto cultured PC12 cells for 48 h. The expressions of neurofilaments (NF68, NF160, and NF200) were determined by specific antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. Quantification plot was shown in histograms. (B) Cultured PC12 cells were treated as in (A) . The cells were fixed with ice-cold 4% paraformaldehyde. The % of differentiated cells (upper panel), the number of neurite per cell (middle panel), and the length of neurite (lower panel) were counted as described in the Materials and Methods section. Bar = 10 μm. NGF (50 ng/mL) served as a positive control. Representative images were shown. Values are expressed as % of total cells in 100 counted cells, Mean ± SEM, n = 4. * p
    Figure Legend Snippet: KXS 2012 does not induce neuronal differentiation. (A) KXS 2012 (5–25 μg/mL) were applied onto cultured PC12 cells for 48 h. The expressions of neurofilaments (NF68, NF160, and NF200) were determined by specific antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. Quantification plot was shown in histograms. (B) Cultured PC12 cells were treated as in (A) . The cells were fixed with ice-cold 4% paraformaldehyde. The % of differentiated cells (upper panel), the number of neurite per cell (middle panel), and the length of neurite (lower panel) were counted as described in the Materials and Methods section. Bar = 10 μm. NGF (50 ng/mL) served as a positive control. Representative images were shown. Values are expressed as % of total cells in 100 counted cells, Mean ± SEM, n = 4. * p

    Techniques Used: Cell Culture, Positive Control

    KXS 2012 increases the expression of TrkA. (A) Cultured PC12 cells were treated with KXS 2012 (15 μg/mL) for 48 h. The cell lysates were collected to determine the expression of Trk A. GAPDH served as a loading control. Quantification plot was shown in right panel. (B) Cultured PC12 cells were treated with KXS 2012 (5–25 μg/mL) for 24 h. The cell lysates were collected to determine the mRNA level of Trk A. Quantification plot was shown. Values are expressed as percentage of increase, where control value is set as 0, Mean ± SEM, n = 4. * p
    Figure Legend Snippet: KXS 2012 increases the expression of TrkA. (A) Cultured PC12 cells were treated with KXS 2012 (15 μg/mL) for 48 h. The cell lysates were collected to determine the expression of Trk A. GAPDH served as a loading control. Quantification plot was shown in right panel. (B) Cultured PC12 cells were treated with KXS 2012 (5–25 μg/mL) for 24 h. The cell lysates were collected to determine the mRNA level of Trk A. Quantification plot was shown. Values are expressed as percentage of increase, where control value is set as 0, Mean ± SEM, n = 4. * p

    Techniques Used: Expressing, Cell Culture

    8) Product Images from "Social defeat stress before pregnancy induces depressive-like behaviours and cognitive deficits in adult male offspring: correlation with neurobiological changes"

    Article Title: Social defeat stress before pregnancy induces depressive-like behaviours and cognitive deficits in adult male offspring: correlation with neurobiological changes

    Journal: BMC Neuroscience

    doi: 10.1186/s12868-018-0463-7

    a Relative levels of BDNF/GAPDH in the hippocampus and the prefrontal cortex of stress and control offspring. b Relative levels of CREB/GAPDH in the hippocampus and the prefrontal cortex. c Relative levels of pCREB/GAPDH in the hippocampus and the prefrontal cortex. d Relative levels of SERT/GAPDH in the hippocampus and the prefrontal cortex. Control offspring (also CP): offspring of control group (black, n = 4); stress offspring (also SP): offspring of maternal rats subject to SDS stimulation (grey, n = 4); BDNF: brain derived neurotrophic factor; CREB: cAMP-response element binding protein; pCREB: phosphorylated cAMP-response element binding protein; SERT: serotonin transporter; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *p
    Figure Legend Snippet: a Relative levels of BDNF/GAPDH in the hippocampus and the prefrontal cortex of stress and control offspring. b Relative levels of CREB/GAPDH in the hippocampus and the prefrontal cortex. c Relative levels of pCREB/GAPDH in the hippocampus and the prefrontal cortex. d Relative levels of SERT/GAPDH in the hippocampus and the prefrontal cortex. Control offspring (also CP): offspring of control group (black, n = 4); stress offspring (also SP): offspring of maternal rats subject to SDS stimulation (grey, n = 4); BDNF: brain derived neurotrophic factor; CREB: cAMP-response element binding protein; pCREB: phosphorylated cAMP-response element binding protein; SERT: serotonin transporter; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *p

    Techniques Used: Derivative Assay, Binding Assay

    9) Product Images from "Signal transducer and activator of transcription 3 signaling upregulates fascin via nuclear factor-?B in gastric cancer: Implications in cell invasion and migration"

    Article Title: Signal transducer and activator of transcription 3 signaling upregulates fascin via nuclear factor-?B in gastric cancer: Implications in cell invasion and migration

    Journal: Oncology Letters

    doi: 10.3892/ol.2014.1804

    STAT3 is required for IL-6-induced expression of fascin and cell migration in MKN45 cells. (A) MKN45 cells were transfected with STAT3 siRNA or treated with AG490 and cultured for 24 h. Whole cell extracts were subjected to western blotting with antibodies against pSTAT3, total STAT3, MMP-2 and -9 and GAPDH. MKN45 cells were treated with IL-6 for 30 min. (B) MKN45 cells were transfected with STAT3 siRNA or treated with AG490 and cultured for 3 days prior to treatment with IL-6 for 30 min. mRNA levels of fascin were detected by quantitative polymerase chain reaction. Results were standardized to GAPDH and expressed as fold induction of IL-6-treated cells from three independent experiments. (C) MKN45 cells were treated as described and wound healing assays were performed. The percentage of the wound occupied in three independent experiments was calculated. (D) MKN45 cells were treated as described and cell migration assays were performed. The mean number of migrated cells in at least 5 visual fields of 3 independent experiments was calculated. (E) MKN45 cells were treated as described and cell invasion assays were performed. Invaded cells were stained and eluted for absorbance readings at 560 nm. (F) Representative experiments of wound healing assays are shown (magnification, ×100). (G) Representative experiments of cell migration assays are shown (magnification, ×100). * P
    Figure Legend Snippet: STAT3 is required for IL-6-induced expression of fascin and cell migration in MKN45 cells. (A) MKN45 cells were transfected with STAT3 siRNA or treated with AG490 and cultured for 24 h. Whole cell extracts were subjected to western blotting with antibodies against pSTAT3, total STAT3, MMP-2 and -9 and GAPDH. MKN45 cells were treated with IL-6 for 30 min. (B) MKN45 cells were transfected with STAT3 siRNA or treated with AG490 and cultured for 3 days prior to treatment with IL-6 for 30 min. mRNA levels of fascin were detected by quantitative polymerase chain reaction. Results were standardized to GAPDH and expressed as fold induction of IL-6-treated cells from three independent experiments. (C) MKN45 cells were treated as described and wound healing assays were performed. The percentage of the wound occupied in three independent experiments was calculated. (D) MKN45 cells were treated as described and cell migration assays were performed. The mean number of migrated cells in at least 5 visual fields of 3 independent experiments was calculated. (E) MKN45 cells were treated as described and cell invasion assays were performed. Invaded cells were stained and eluted for absorbance readings at 560 nm. (F) Representative experiments of wound healing assays are shown (magnification, ×100). (G) Representative experiments of cell migration assays are shown (magnification, ×100). * P

    Techniques Used: Expressing, Migration, Transfection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Staining

    10) Product Images from "Expression and potential role of SNF5 in endometrial carcinoma"

    Article Title: Expression and potential role of SNF5 in endometrial carcinoma

    Journal: BMC Women's Health

    doi: 10.1186/s12905-019-0718-1

    Western blotting analysis. Ishikawa cells were collected after transfection with two concentration siRNA (25 or 50 nM) against SNF5 or control siRNA. ( a ) Precipitated proteins were measured by western blotting using antibodies targeting SNF5 or p21. GAPDH was used as an internal control. ( b ) The western blotting results were analysed by Mann-Whitney test using GraphPad 6.0 software. Data are shown as the mean ± SD of three independent experiments, * P
    Figure Legend Snippet: Western blotting analysis. Ishikawa cells were collected after transfection with two concentration siRNA (25 or 50 nM) against SNF5 or control siRNA. ( a ) Precipitated proteins were measured by western blotting using antibodies targeting SNF5 or p21. GAPDH was used as an internal control. ( b ) The western blotting results were analysed by Mann-Whitney test using GraphPad 6.0 software. Data are shown as the mean ± SD of three independent experiments, * P

    Techniques Used: Western Blot, Transfection, Concentration Assay, MANN-WHITNEY, Software

    11) Product Images from "Divergent Effects of PERK and IRE1 Signaling on Cell Viability"

    Article Title: Divergent Effects of PERK and IRE1 Signaling on Cell Viability

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004170

    Selective and specific activation of IRE1 signaling. (A) Parental wild-type and transgenic HEK293 cells expressing the Ire1[I642G] , or Ire1[I642G/K599A] allele were treated for the indicated times with 1NM-PP1 (1 µM), and wild-type cells were treated for 4 hours with thapsigargin (tg) (300 nM). IRE1[I642G] and IRE1[I642G/K599A] protein was detected by immunoblotting for the FLAG epitope. GAPDH levels were assessed as a protein loading control. Xbp1 mRNA splicing was determined by RT-PCR. The unspliced ( u ) and spliced ( s ) Xbp1 mRNA products are indicated as labeled. ERdj4 mRNA levels were measured by quantitative PCR, normalized to Rpl19 mRNA levels, and are shown relative to levels in untreated cells. (B) Parental wild-type and transgenic HEK293 cells expressing the Ire1[I642G] or Ire1[I642G/K599A] alleles were treated for the indicated times with 1NM-PP1 (1 µM); wild-type cells were also treated for 4 hours with thapsigargin (tg) (300 nM). ATF4 protein was detected by immunoblotting. GAPDH levels were assessed as a protein loading control.
    Figure Legend Snippet: Selective and specific activation of IRE1 signaling. (A) Parental wild-type and transgenic HEK293 cells expressing the Ire1[I642G] , or Ire1[I642G/K599A] allele were treated for the indicated times with 1NM-PP1 (1 µM), and wild-type cells were treated for 4 hours with thapsigargin (tg) (300 nM). IRE1[I642G] and IRE1[I642G/K599A] protein was detected by immunoblotting for the FLAG epitope. GAPDH levels were assessed as a protein loading control. Xbp1 mRNA splicing was determined by RT-PCR. The unspliced ( u ) and spliced ( s ) Xbp1 mRNA products are indicated as labeled. ERdj4 mRNA levels were measured by quantitative PCR, normalized to Rpl19 mRNA levels, and are shown relative to levels in untreated cells. (B) Parental wild-type and transgenic HEK293 cells expressing the Ire1[I642G] or Ire1[I642G/K599A] alleles were treated for the indicated times with 1NM-PP1 (1 µM); wild-type cells were also treated for 4 hours with thapsigargin (tg) (300 nM). ATF4 protein was detected by immunoblotting. GAPDH levels were assessed as a protein loading control.

    Techniques Used: Activation Assay, Transgenic Assay, Expressing, FLAG-tag, Reverse Transcription Polymerase Chain Reaction, Labeling, Real-time Polymerase Chain Reaction

    12) Product Images from "Epigenetic Upregulation of Urokinase Plasminogen Activator Promotes the Tropism of Mesenchymal Stem Cells for Tumor Cells"

    Article Title: Epigenetic Upregulation of Urokinase Plasminogen Activator Promotes the Tropism of Mesenchymal Stem Cells for Tumor Cells

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-09-0495

    Expression levels of the uPA and uPAR in human tumor and non-tumor cell lines (A) uPA and uPAR mRNA expression in RWPE1, HEK293, U251, MDA231 and PC3 cells were analyzed by quantitative RT-PCR. (B) Immunoblot analysis of uPA and uPAR proteins in HEK293, U251, MDA231 and PC3 cells. GAPDH was used as a loading control.
    Figure Legend Snippet: Expression levels of the uPA and uPAR in human tumor and non-tumor cell lines (A) uPA and uPAR mRNA expression in RWPE1, HEK293, U251, MDA231 and PC3 cells were analyzed by quantitative RT-PCR. (B) Immunoblot analysis of uPA and uPAR proteins in HEK293, U251, MDA231 and PC3 cells. GAPDH was used as a loading control.

    Techniques Used: Expressing, Quantitative RT-PCR

    Overexpression of uPA enhances the tumor-specific migration ability of MSCs (A) RT-PCR ( top ) and immunoblot ( bottom ) analyses of CB-MSCs stably transduced with mock, uPA expression vector or empty vector. GAPDH was used as loading control for RNA and protein analysis. (B) The migration capacity of the uPA-overexpressing MSCs toward PC3 cells was assessed in vitro by transwell migration assay. Columns, percentage of the vector control (* p
    Figure Legend Snippet: Overexpression of uPA enhances the tumor-specific migration ability of MSCs (A) RT-PCR ( top ) and immunoblot ( bottom ) analyses of CB-MSCs stably transduced with mock, uPA expression vector or empty vector. GAPDH was used as loading control for RNA and protein analysis. (B) The migration capacity of the uPA-overexpressing MSCs toward PC3 cells was assessed in vitro by transwell migration assay. Columns, percentage of the vector control (* p

    Techniques Used: Over Expression, Migration, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transduction, Expressing, Plasmid Preparation, In Vitro, Transwell Migration Assay

    Activation of ERK by uPA mediates the tumor-specific migration capacity of MSCs (A) Immunoblot analysis shows increased levels of ERK1/2 phosphorylation in uPA-expressing MSCs (uPA-10). GAPDH was used as a loading control. (B) Pretreatment of ERK inhibitor significantly decreased uPA-expressing MSC migration toward PC3 and MDA231 cells compared with cells treated with nonspecific isotype IgG. Columns, percentage of the vector control (* p
    Figure Legend Snippet: Activation of ERK by uPA mediates the tumor-specific migration capacity of MSCs (A) Immunoblot analysis shows increased levels of ERK1/2 phosphorylation in uPA-expressing MSCs (uPA-10). GAPDH was used as a loading control. (B) Pretreatment of ERK inhibitor significantly decreased uPA-expressing MSC migration toward PC3 and MDA231 cells compared with cells treated with nonspecific isotype IgG. Columns, percentage of the vector control (* p

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

    13) Product Images from "Retention of Mitochondria in Mature Human Red Blood Cells as the Result of Autophagy Impairment in Rett Syndrome"

    Article Title: Retention of Mitochondria in Mature Human Red Blood Cells as the Result of Autophagy Impairment in Rett Syndrome

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12069-0

    Defective autophagy activation in RTT fibroblasts. ( a ) WB analysis of RTT and healthy fibroblasts cultivated either in DMEM or starvation medium (2 h) in the presence or absence of 20 µM of CQ ( upper panel ). Filters were probed with an anti-LC3B or an anti-GAPDH antibody. A representative immunoblot of three independent experiments performed on the cell lines available (n = 4 for both the healthy and the RTT fibroblasts) is reported. Densitometric determination of LC3B to GAPDH content was performed by ImageJ software ( lower panel ). Results are the means ± S.E. of three independent experiments. Differences between the different experimental conditions in the same group are significantly different (* p
    Figure Legend Snippet: Defective autophagy activation in RTT fibroblasts. ( a ) WB analysis of RTT and healthy fibroblasts cultivated either in DMEM or starvation medium (2 h) in the presence or absence of 20 µM of CQ ( upper panel ). Filters were probed with an anti-LC3B or an anti-GAPDH antibody. A representative immunoblot of three independent experiments performed on the cell lines available (n = 4 for both the healthy and the RTT fibroblasts) is reported. Densitometric determination of LC3B to GAPDH content was performed by ImageJ software ( lower panel ). Results are the means ± S.E. of three independent experiments. Differences between the different experimental conditions in the same group are significantly different (* p

    Techniques Used: Activation Assay, Western Blot, Software

    14) Product Images from "Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA"

    Article Title: Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.056176

    Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P
    Figure Legend Snippet: Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P

    Techniques Used: Transfection, Lysis, Incubation, In Vitro, Synthesized, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    15) Product Images from "Lin28b Promotes Head and Neck Cancer Progression via Modulation of the Insulin-Like Growth Factor Survival Pathway"

    Article Title: Lin28b Promotes Head and Neck Cancer Progression via Modulation of the Insulin-Like Growth Factor Survival Pathway

    Journal: Oncotarget

    doi:

    Stable expression of Lin28b enhanced HNSCC tumorigenicity in vitro and in vivo Expression of Lin28b in FaDu cells stably transfected with Lin28b-expression vector was detected by (A) qRT-PCR; and (B) Western blotting (27 kd), using GAPDH (37 kd) for loading control. (C) Representative images depicting enhanced migration ability of FaDu~Lin28b cells compared FaDu~GFP cells. Data were presented as mean ± S.E, n=10. (D) FaDu~Lin28b cells exhibited enhanced tumor formation and radiation resistance in vivo . Data were presented as mean ± S.E, n=4-5 mice/group. **p
    Figure Legend Snippet: Stable expression of Lin28b enhanced HNSCC tumorigenicity in vitro and in vivo Expression of Lin28b in FaDu cells stably transfected with Lin28b-expression vector was detected by (A) qRT-PCR; and (B) Western blotting (27 kd), using GAPDH (37 kd) for loading control. (C) Representative images depicting enhanced migration ability of FaDu~Lin28b cells compared FaDu~GFP cells. Data were presented as mean ± S.E, n=10. (D) FaDu~Lin28b cells exhibited enhanced tumor formation and radiation resistance in vivo . Data were presented as mean ± S.E, n=4-5 mice/group. **p

    Techniques Used: Expressing, In Vitro, In Vivo, Stable Transfection, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Migration, Mouse Assay

    16) Product Images from "Lactobacillus salivarius alleviates inflammation via NF-κB signaling in ETEC K88-induced IPEC-J2 cells"

    Article Title: Lactobacillus salivarius alleviates inflammation via NF-κB signaling in ETEC K88-induced IPEC-J2 cells

    Journal: Journal of Animal Science and Biotechnology

    doi: 10.1186/s40104-020-00488-5

    Activation of MAPK and NF-κB pathways in ETEC K88-stimulated IPEC-J2 cells. IPEC-J2 cells harvested from L. salivarius involvement groups and control ones were treated with ETEC K88, and cell lysates were subjected to Western blotting with phosphor-specific Abs against ( a ) p-ERK, ( b ) p-p38 and ( c ) p-p65. GAPDH was used as the loading control. Data are presented as means ± SEM of three independent experiments. *, P
    Figure Legend Snippet: Activation of MAPK and NF-κB pathways in ETEC K88-stimulated IPEC-J2 cells. IPEC-J2 cells harvested from L. salivarius involvement groups and control ones were treated with ETEC K88, and cell lysates were subjected to Western blotting with phosphor-specific Abs against ( a ) p-ERK, ( b ) p-p38 and ( c ) p-p65. GAPDH was used as the loading control. Data are presented as means ± SEM of three independent experiments. *, P

    Techniques Used: Activation Assay, Western Blot

    17) Product Images from "Reversion of resistance to oxaliplatin by inhibition of p38 MAPK in colorectal cancer cell lines: involvement of the calpain / Nox1 pathway"

    Article Title: Reversion of resistance to oxaliplatin by inhibition of p38 MAPK in colorectal cancer cell lines: involvement of the calpain / Nox1 pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21780

    Regulation of Nox1 by calpain HT29-D4, Rox1 and Rox2 cells were transfected with control siRNA (si Control), calpain 1 specific siRNA (si Calpain 1), calpain 2 specific siRNA (si Calpain 2) or with both siRNA (si Calpain 1/2). The cells were seeded in white 96-well plates to perform lucigenin assays (A) , and in 6-well plates to perform Western blots after oxaliplatin treatment (2 μM) (C) . HT29-D4, Rox1 and Rox2 cells were transfected with control siRNA (si CTRL) or PKC d specific siRNA (si PKC delta) and seeded in white 96-well plates to perform lucigenin assays (B) . NoxA1 was immunoprecipitated from HT29-D4 lysates and incubated with purified calpain 1. The samples were then processed for immunoblotting using antibodies against calpain 1, NoxA1 and GAPDH (Control: total lysate before IP, (D) . HT29-D4 cells were transfected with a plasmid encoding NoxA1-DDK. The cells were lysed, and NoxA1 was immunoprecipitated (IP NoxA1) and incubated with purified calpain 1. The samples were then processed for immunoblotting using antibodies against calpain 1, DDK tag and GAPDH (E) . HT29-D4, Rox1 and Rox2 cells were seeded in white 96-well plates to perform lucigenin assays in the absence (Control) or in the presence of Src inhibitor ( F ). HT29-D4 (HT29), Rox1 and Rox2 cells were transfected with control siRNA (si Control), or both calpain 1 and calpain 2 specific siRNAs (si Calpain 1/2). The cells were then lysed and the proteins were processed for immunoblotting using antibodies against Src, p-Src (tyrosine 527) and GAPDH (G) . Asteriks indicate a statistical significance with p
    Figure Legend Snippet: Regulation of Nox1 by calpain HT29-D4, Rox1 and Rox2 cells were transfected with control siRNA (si Control), calpain 1 specific siRNA (si Calpain 1), calpain 2 specific siRNA (si Calpain 2) or with both siRNA (si Calpain 1/2). The cells were seeded in white 96-well plates to perform lucigenin assays (A) , and in 6-well plates to perform Western blots after oxaliplatin treatment (2 μM) (C) . HT29-D4, Rox1 and Rox2 cells were transfected with control siRNA (si CTRL) or PKC d specific siRNA (si PKC delta) and seeded in white 96-well plates to perform lucigenin assays (B) . NoxA1 was immunoprecipitated from HT29-D4 lysates and incubated with purified calpain 1. The samples were then processed for immunoblotting using antibodies against calpain 1, NoxA1 and GAPDH (Control: total lysate before IP, (D) . HT29-D4 cells were transfected with a plasmid encoding NoxA1-DDK. The cells were lysed, and NoxA1 was immunoprecipitated (IP NoxA1) and incubated with purified calpain 1. The samples were then processed for immunoblotting using antibodies against calpain 1, DDK tag and GAPDH (E) . HT29-D4, Rox1 and Rox2 cells were seeded in white 96-well plates to perform lucigenin assays in the absence (Control) or in the presence of Src inhibitor ( F ). HT29-D4 (HT29), Rox1 and Rox2 cells were transfected with control siRNA (si Control), or both calpain 1 and calpain 2 specific siRNAs (si Calpain 1/2). The cells were then lysed and the proteins were processed for immunoblotting using antibodies against Src, p-Src (tyrosine 527) and GAPDH (G) . Asteriks indicate a statistical significance with p

    Techniques Used: Transfection, Western Blot, Immunoprecipitation, Incubation, Purification, Plasmid Preparation

    Implication of Nox1 in oxaliplatin-induced ROS production and cytotoxicity The effects of oxaliplatin on cell viability were studied with HT29-D4, Rox1 and Rox2 cells treated with apocynin (A) or transfected with control siRNA (si Control) or Nox1 specific siRNA (si Nox1) (B) . HT29-D4, Rox1 and Rox2 cells were lysed, and equal amounts of cellular proteins were processed for immunoblotting using the antibodies against Nox1, NoxA1, NoxO1 and GAPDH (C) . HT29-D4, Rox1 and Rox2 cells were transfected with control siRNA (si Control) and Nox1 specific siRNA (si Nox1). The cells were lysed, and equal amounts of cellular protein were processed for immunoblotting using the antibodies against Nox1 (D) . Transfected cells were also seeded in white 96-well plates to perform lucigenin assays ( E ) and in black 96-well plates to perform Amplex red assay (F) . These cells were treated with 2 μM oxaliplatin over time (- untreated, 45 minutes (45 min), 4 hours (4h) and 24 hours (24h)). Asteriks indicate a statistical significance with p
    Figure Legend Snippet: Implication of Nox1 in oxaliplatin-induced ROS production and cytotoxicity The effects of oxaliplatin on cell viability were studied with HT29-D4, Rox1 and Rox2 cells treated with apocynin (A) or transfected with control siRNA (si Control) or Nox1 specific siRNA (si Nox1) (B) . HT29-D4, Rox1 and Rox2 cells were lysed, and equal amounts of cellular proteins were processed for immunoblotting using the antibodies against Nox1, NoxA1, NoxO1 and GAPDH (C) . HT29-D4, Rox1 and Rox2 cells were transfected with control siRNA (si Control) and Nox1 specific siRNA (si Nox1). The cells were lysed, and equal amounts of cellular protein were processed for immunoblotting using the antibodies against Nox1 (D) . Transfected cells were also seeded in white 96-well plates to perform lucigenin assays ( E ) and in black 96-well plates to perform Amplex red assay (F) . These cells were treated with 2 μM oxaliplatin over time (- untreated, 45 minutes (45 min), 4 hours (4h) and 24 hours (24h)). Asteriks indicate a statistical significance with p

    Techniques Used: Transfection, Amplex Red Assay

    18) Product Images from "Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation"

    Article Title: Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation

    Journal: Scientific Reports

    doi: 10.1038/srep33897

    LRRK2 kinase inhibition reduces LRRK2 protein levels. SH-SY5Y overexpressing 3flag-LRRK2 WT ( a ) A2016T ( b ) K1906M ( c ) G2019S ( d ) S910A ( e ) or S935A ( f ) were treated according to different time schedules with LRRK2-IN1 (L2-IN1, 1 μM), CZC-25146 (CZC, 200 nM), PF-06447475 (PF, 150 nM), GSK2578215A (GSK, 1 μM), MLi-2 (10 nM) or HG 10-102-01 (HG, 1 μM) or DMSO. Cell lysates were analyzed with immunoblotting using FlagM2 antibody for LRRK2 detection, anti-LRRK2 P-S935 and anti-α- or β-tubulin, anti-GAPDH or anti-vinculin for equal loading. Shown are representative blots. Graphs show the quantification of blots representing the ratio of phosphorylation at S935 over total LRRK2 signal or total LRRK2 over housekeeping protein signal. Error bars indicate s.e.m. with N ≥ 3. Statistical significance was tested using a two-way ANOVA test with Bonferroni post-tests. ***p
    Figure Legend Snippet: LRRK2 kinase inhibition reduces LRRK2 protein levels. SH-SY5Y overexpressing 3flag-LRRK2 WT ( a ) A2016T ( b ) K1906M ( c ) G2019S ( d ) S910A ( e ) or S935A ( f ) were treated according to different time schedules with LRRK2-IN1 (L2-IN1, 1 μM), CZC-25146 (CZC, 200 nM), PF-06447475 (PF, 150 nM), GSK2578215A (GSK, 1 μM), MLi-2 (10 nM) or HG 10-102-01 (HG, 1 μM) or DMSO. Cell lysates were analyzed with immunoblotting using FlagM2 antibody for LRRK2 detection, anti-LRRK2 P-S935 and anti-α- or β-tubulin, anti-GAPDH or anti-vinculin for equal loading. Shown are representative blots. Graphs show the quantification of blots representing the ratio of phosphorylation at S935 over total LRRK2 signal or total LRRK2 over housekeeping protein signal. Error bars indicate s.e.m. with N ≥ 3. Statistical significance was tested using a two-way ANOVA test with Bonferroni post-tests. ***p

    Techniques Used: Inhibition

    19) Product Images from "RNA:DNA hybrids are a novel molecular pattern sensed by TLR9"

    Article Title: RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

    Journal: The EMBO Journal

    doi: 10.1002/embj.201386117

    Viral RNA:DNA hybrids accumulate in the cytoplasm and endosomes of MMLV infected cells. A, B Viral RNA:DNA hybrids are present in the cytoplasm of MMLV infected cells. (A) MMLV-specific PCR detects RNA:DNA hybrids immunoprecipitated from cytoplasmic nucleic acids. As PCR detects both MMLV DNA and RNA:DNA hybrids, specificity of the S9.6 pull down for hybrids is confirmed by the absence of PCR product in control samples processed with either S9.6 antibody omitted or when cytoplasmic nucleic acids were pretreated with RNase H. Labels: H 2 O, PCR without template added; Input, cytoplasmic nucleic acids prior to immunopreciptation (equal in proportion to post-pull down template); Uninfected, cytoplasmic nucleic acids from uninfected B3T3 cells. B Quantification of MMLV RNA:DNA hybrids immunoprecipitated by S9.6 by qPCR using two independent MMLV primer sets. S9.6 immunoprecipitate contains 4.1 ± 1.1% of input molecules. RNase H treatment reduces the amount of MMLV-DNA pulled down by S9.6 by > 90%. Data shown are the mean of three independent IP/qPCR experiments ± s.d. * P = 0.0366 (MMLV-1), * P = 0.231 (MMLV-2). C Validation of endosomal fractionation: the early endosome marker Rab5 is enriched in endosomal preparations. Western blotting of cytoplasmic and endosomal fractions shows the presence of the endosomal marker Rab5 in both endosomal and cytoplasmic fractions, whereas GAPDH is only present in cytoplasmic fractions. Densitometry measurements show that relative Rab5 enrichment is > 22-fold relative to GAPDH. D Viral RNA:DNA hybrids are present in endosomal fractions of MMLV-infected cells. MMLV DNA was detected by PCR after S9.6 pull-down of hybrids from endosomal nucleic acids, but not in beads only or RNase H treated controls.
    Figure Legend Snippet: Viral RNA:DNA hybrids accumulate in the cytoplasm and endosomes of MMLV infected cells. A, B Viral RNA:DNA hybrids are present in the cytoplasm of MMLV infected cells. (A) MMLV-specific PCR detects RNA:DNA hybrids immunoprecipitated from cytoplasmic nucleic acids. As PCR detects both MMLV DNA and RNA:DNA hybrids, specificity of the S9.6 pull down for hybrids is confirmed by the absence of PCR product in control samples processed with either S9.6 antibody omitted or when cytoplasmic nucleic acids were pretreated with RNase H. Labels: H 2 O, PCR without template added; Input, cytoplasmic nucleic acids prior to immunopreciptation (equal in proportion to post-pull down template); Uninfected, cytoplasmic nucleic acids from uninfected B3T3 cells. B Quantification of MMLV RNA:DNA hybrids immunoprecipitated by S9.6 by qPCR using two independent MMLV primer sets. S9.6 immunoprecipitate contains 4.1 ± 1.1% of input molecules. RNase H treatment reduces the amount of MMLV-DNA pulled down by S9.6 by > 90%. Data shown are the mean of three independent IP/qPCR experiments ± s.d. * P = 0.0366 (MMLV-1), * P = 0.231 (MMLV-2). C Validation of endosomal fractionation: the early endosome marker Rab5 is enriched in endosomal preparations. Western blotting of cytoplasmic and endosomal fractions shows the presence of the endosomal marker Rab5 in both endosomal and cytoplasmic fractions, whereas GAPDH is only present in cytoplasmic fractions. Densitometry measurements show that relative Rab5 enrichment is > 22-fold relative to GAPDH. D Viral RNA:DNA hybrids are present in endosomal fractions of MMLV-infected cells. MMLV DNA was detected by PCR after S9.6 pull-down of hybrids from endosomal nucleic acids, but not in beads only or RNase H treated controls.

    Techniques Used: Infection, Polymerase Chain Reaction, Immunoprecipitation, Real-time Polymerase Chain Reaction, Fractionation, Marker, Western Blot

    20) Product Images from "miR-218 Inhibits Erythroid Differentiation and Alters Iron Metabolism by Targeting ALAS2 in K562 Cells"

    Article Title: miR-218 Inhibits Erythroid Differentiation and Alters Iron Metabolism by Targeting ALAS2 in K562 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms161226088

    ( A ) Relative mature miRNA expression in K562 control cells and each cell line overexpressing one of the candidate miRNAs using quantitative real-time PCR normalized to snRNA U6 ; ( B ) Relative expression of ALAS2 in control and each miRNA overexpression K562 cells using quantitative real-time PCR normalized to GAPDH ; ( C ) K562 cells were transfected with candidate miRNA (or pcDNA3.0 as control) and the luciferase construct with the ALAS2-3′UTR. After 48 h, relative firefly/renilla luciferase activity in the cells was evaluated. Co-transfection of miR-124 and vimentin ( VIM ) 3′UTR was performed as a positive control [ 28 ] as shown in the second column. Asterisks indicate that differences between samples were statistically significant according to an independent-sample t -test, * p
    Figure Legend Snippet: ( A ) Relative mature miRNA expression in K562 control cells and each cell line overexpressing one of the candidate miRNAs using quantitative real-time PCR normalized to snRNA U6 ; ( B ) Relative expression of ALAS2 in control and each miRNA overexpression K562 cells using quantitative real-time PCR normalized to GAPDH ; ( C ) K562 cells were transfected with candidate miRNA (or pcDNA3.0 as control) and the luciferase construct with the ALAS2-3′UTR. After 48 h, relative firefly/renilla luciferase activity in the cells was evaluated. Co-transfection of miR-124 and vimentin ( VIM ) 3′UTR was performed as a positive control [ 28 ] as shown in the second column. Asterisks indicate that differences between samples were statistically significant according to an independent-sample t -test, * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Luciferase, Construct, Activity Assay, Cotransfection, Positive Control

    ( A ) Relative mRNA expression of ALAS2 in K562 control and K562 ALAS2-sh cells using quantitative real-time PCR; ( B ) Representative Western blots showing ALAS2 expression in K562 control and K562 ALAS2-sh cells. Quantification normalized to GAPDH by densitometry using ImageJ was provided; ( C ) Hemoglobin concentration of K562 control and K562 ALAS2-sh cells. Cell surface expression of CD235a ( D ) and mRNA levels ( E ) of K562 control (black line in D ) and K562 ALAS2-sh using FACS and quantitative real-time PCR, respectively; ( F ) Relative mRNA expression of IRP2 and TFR1 in K562 control and K562 ALAS2-sh cells using quantitative real-time PCR. All relative mRNA expression using quantitative real-time PCR was normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) expression. Asterisks indicate that differences between samples were statistically significant according to an independent-sample t -test, * p
    Figure Legend Snippet: ( A ) Relative mRNA expression of ALAS2 in K562 control and K562 ALAS2-sh cells using quantitative real-time PCR; ( B ) Representative Western blots showing ALAS2 expression in K562 control and K562 ALAS2-sh cells. Quantification normalized to GAPDH by densitometry using ImageJ was provided; ( C ) Hemoglobin concentration of K562 control and K562 ALAS2-sh cells. Cell surface expression of CD235a ( D ) and mRNA levels ( E ) of K562 control (black line in D ) and K562 ALAS2-sh using FACS and quantitative real-time PCR, respectively; ( F ) Relative mRNA expression of IRP2 and TFR1 in K562 control and K562 ALAS2-sh cells using quantitative real-time PCR. All relative mRNA expression using quantitative real-time PCR was normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) expression. Asterisks indicate that differences between samples were statistically significant according to an independent-sample t -test, * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay, FACS

    ( A ) The overexpression of miR-218 in K562 cells was confirmed using mature miRNA quantitative real-time PCR normalized to U6 . The expression of ALAS2 at the mRNA level ( B ) and at the protein level ( C ) in K562 control and K562 miR-218 cells was measured by quantitative real-time PCR and western blot, respectively. Western blot quantification normalized to GAPDH by densitometry using ImageJ was provided; ( D ) Predicted binding site for the seed sequence of miR-218 in the 3′UTR of ALAS2 mRNA. Four mutated forms of the 3′UTR were used to generate luciferase reporter constructs as shown. The mutated nucleic acid was shown in bold and underlines; ( E ) miR-218 inhibits wild-type, but not the mutated ALAS2 3′UTR. Asterisks indicate that differences between samples were statistically significant according to an independent-sample t -test, * p
    Figure Legend Snippet: ( A ) The overexpression of miR-218 in K562 cells was confirmed using mature miRNA quantitative real-time PCR normalized to U6 . The expression of ALAS2 at the mRNA level ( B ) and at the protein level ( C ) in K562 control and K562 miR-218 cells was measured by quantitative real-time PCR and western blot, respectively. Western blot quantification normalized to GAPDH by densitometry using ImageJ was provided; ( D ) Predicted binding site for the seed sequence of miR-218 in the 3′UTR of ALAS2 mRNA. Four mutated forms of the 3′UTR were used to generate luciferase reporter constructs as shown. The mutated nucleic acid was shown in bold and underlines; ( E ) miR-218 inhibits wild-type, but not the mutated ALAS2 3′UTR. Asterisks indicate that differences between samples were statistically significant according to an independent-sample t -test, * p

    Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Binding Assay, Sequencing, Luciferase, Construct

    21) Product Images from "A Chinese Herbal Preparation, Xiao-Er-An-Shen Decoction, Exerts Neuron Protection by Modulation of Differentiation and Antioxidant Activity in Cultured PC12 Cells"

    Article Title: A Chinese Herbal Preparation, Xiao-Er-An-Shen Decoction, Exerts Neuron Protection by Modulation of Differentiation and Antioxidant Activity in Cultured PC12 Cells

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2018/8670421

    XEASD stimulates neurofilament expression of cultured PC12 cells. (a) XEASD extracts (0.3–3.0 mg/mL) were applied onto cultured PC12 cells for 48 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. NGF (50 ng/mL) served as the positive control. GAPDH served as a loading control. (b) Quantification plot was shown in histograms. Values are expressed as the fold of increase to basal reading (untreated culture, set as 1). Mean ± SEM; n = 4. Statistical comparison was made with the control; ∗ p
    Figure Legend Snippet: XEASD stimulates neurofilament expression of cultured PC12 cells. (a) XEASD extracts (0.3–3.0 mg/mL) were applied onto cultured PC12 cells for 48 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. NGF (50 ng/mL) served as the positive control. GAPDH served as a loading control. (b) Quantification plot was shown in histograms. Values are expressed as the fold of increase to basal reading (untreated culture, set as 1). Mean ± SEM; n = 4. Statistical comparison was made with the control; ∗ p

    Techniques Used: Expressing, Cell Culture, Positive Control

    22) Product Images from "MAP Kinase-Mediated c-fos Regulation Relies on a Histone Acetylation Relay Switch"

    Article Title: MAP Kinase-Mediated c-fos Regulation Relies on a Histone Acetylation Relay Switch

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2008.01.019

    p300-Mediated Histone Acetylation Promotes Recruitment of NFI to the c -fos Promoter (A) ChIP showing the association of NFI, Elk-1, and acetylated histone H4 with the c - fos promoter upon TSA treatment. HeLa cells were serum starved (−) or starved and treated with TSA for 30 min (+) where indicated. (B) Immunoblot showing the knockdown of p300 protein expression in HeLa cells 48 hr after transfecting with siRNAs targeting p300. (C) ChIP of the c -fos promoter with either an antibody directed toward NFI or normal rabbit IgG. HeLa cells were transfected with siRNA directed toward either p300 or GAPDH and were treated with PMA for 10 min. (D and E) Chromatin accessibility by real-time PCR (CHART-PCR) at the c -fos promoter. Schematic diagram displays the positions of primers (arrows) used for CHART-PCR analysis. HeLa cells were treated with PMA or TSA for 10 min. Where indicated, cells were pretreated with si-p300 before stimulation. Aliquots of isolated nuclei were incubated with increasing amounts of DNase I (0, 1.0, and 2.0 U), and the relative levels of nuclease protection at the nucleosome positioned at the c -fos promoter were measured by real-time PCR. (F) ChIP of the c -fos promoter with either an antibody directed toward total histone H3 or acetylated histone H3 from serum-starved HeLa cells (−) or PMA-treated cells (10 min) (+) as indicated. Data in (A), (C), (D), (E), and (F) are presented as means ± SEM (n ≥ 4, 4, 6, 6, and 4, respectively) and are the average of at least two ([A], [C], and [F]) or three ([D] and [E]) independent experiments performed in duplicate.
    Figure Legend Snippet: p300-Mediated Histone Acetylation Promotes Recruitment of NFI to the c -fos Promoter (A) ChIP showing the association of NFI, Elk-1, and acetylated histone H4 with the c - fos promoter upon TSA treatment. HeLa cells were serum starved (−) or starved and treated with TSA for 30 min (+) where indicated. (B) Immunoblot showing the knockdown of p300 protein expression in HeLa cells 48 hr after transfecting with siRNAs targeting p300. (C) ChIP of the c -fos promoter with either an antibody directed toward NFI or normal rabbit IgG. HeLa cells were transfected with siRNA directed toward either p300 or GAPDH and were treated with PMA for 10 min. (D and E) Chromatin accessibility by real-time PCR (CHART-PCR) at the c -fos promoter. Schematic diagram displays the positions of primers (arrows) used for CHART-PCR analysis. HeLa cells were treated with PMA or TSA for 10 min. Where indicated, cells were pretreated with si-p300 before stimulation. Aliquots of isolated nuclei were incubated with increasing amounts of DNase I (0, 1.0, and 2.0 U), and the relative levels of nuclease protection at the nucleosome positioned at the c -fos promoter were measured by real-time PCR. (F) ChIP of the c -fos promoter with either an antibody directed toward total histone H3 or acetylated histone H3 from serum-starved HeLa cells (−) or PMA-treated cells (10 min) (+) as indicated. Data in (A), (C), (D), (E), and (F) are presented as means ± SEM (n ≥ 4, 4, 6, 6, and 4, respectively) and are the average of at least two ([A], [C], and [F]) or three ([D] and [E]) independent experiments performed in duplicate.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Transfection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Incubation

    23) Product Images from "Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks"

    Article Title: Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0510764103

    Overexpression of TAF4b in GCs. ( A ) Immunoblotting of total cell extracts from stable f-TAF4b and flag control cell lines was performed to determine levels of TFIID subunits and general transcription factors by using anti-Pol II, anti-TAF1, anti-TAF4, anti-TFIIB, anti-Flag, and an anti-TAF4b monoclonal antibody. Anti-GAPDH antibodies were used to confirm equal loading. ( B ) Nuclear localization of Flag-TAF4b in SIGCs was detected by indirect immunofluorescence using an anti-Flag antibody. Nuclei were counterstained with Hoechst dye to label DNA. Arrows indicate nucleoli. ( C ) Partially purified nuclear extracts from f-TAF4b cells were precipitated with a control and anti-TAF4b polyclonal antibodies, followed by immunoblotting with anti-TAF4, anti-Flag, and anti-TBP antibodies.
    Figure Legend Snippet: Overexpression of TAF4b in GCs. ( A ) Immunoblotting of total cell extracts from stable f-TAF4b and flag control cell lines was performed to determine levels of TFIID subunits and general transcription factors by using anti-Pol II, anti-TAF1, anti-TAF4, anti-TFIIB, anti-Flag, and an anti-TAF4b monoclonal antibody. Anti-GAPDH antibodies were used to confirm equal loading. ( B ) Nuclear localization of Flag-TAF4b in SIGCs was detected by indirect immunofluorescence using an anti-Flag antibody. Nuclei were counterstained with Hoechst dye to label DNA. Arrows indicate nucleoli. ( C ) Partially purified nuclear extracts from f-TAF4b cells were precipitated with a control and anti-TAF4b polyclonal antibodies, followed by immunoblotting with anti-TAF4, anti-Flag, and anti-TBP antibodies.

    Techniques Used: Over Expression, Immunofluorescence, Purification

    24) Product Images from "RING finger protein 31 promotes p53 degradation in breast cancer cells"

    Article Title: RING finger protein 31 promotes p53 degradation in breast cancer cells

    Journal: Oncogene

    doi: 10.1038/onc.2015.260

    RNF31 regulates p53 protein stability. ( a ) Overexpression of RNF31 decreases endogenous p53 protein levels in MCF-7 cells. MCF-7 cells were transfected with plasmids expressing Flag-tagged RNF31 or the Flag tag alone. After 48 h, whole-protein extracts were prepared and the levels of RNF31, p53 and the internal control Glyceraldehydes 3-phophate dehydrogenase (GAPDH) were determined by western blot analysis. ( b ) RNF31 depletion increases p53 protein levels. MCF-7 cells were transfected with siRNF31 or siControl. Cells were collected after 24 h. p53 and RNF31 levels were determined by western blot analysis. GAPDH was used as internal control. ( c ) RNF31 depletion does not further increase the stability of p53 in the presence of the proteasome inhibitor MG132. MCF-7 cells were transfected with siRNF31 or siControl. After 48 h, cells were treated with 10 μ M MG132 or vehicle. Cells were collected 6 h after treatment and whole-protein extracts were prepared. The levels of RNF31, p53 and the internal control GAPDH were determined by western blot analysis. ( d ) Depletion of RNF31 increases p53 protein stability. MCF-7 cells were transfected with siRNF31 or siControl. After 48 h, cells were treated with protein biosynthesis inhibitor (100 μ M cycloheximide) for different times before whole-protein extraction. The levels of RNF31, p53 and the internal control GAPDH were determined by western blot analysis. ImageJ was used to quantify the p53 band density, followed by a normalization of the p53 level, with the level at time point zero set as 1. ( e ) Overexpression of RNF31 decreases p53 protein stability. MCF-7 cells were transfected with siRNF31 or siControl. After 48 h, cells were treated with protein biosynthesis inhibitor (100 μ M cycloheximide) for different times before whole-protein extraction. The levels of RNF31, p53 and the internal control GAPDH were determined by western blot analysis. ImageJ was used to quantify the p53 band density, followed by a normalization of the p53 level, with the level at time point zero set as 1.
    Figure Legend Snippet: RNF31 regulates p53 protein stability. ( a ) Overexpression of RNF31 decreases endogenous p53 protein levels in MCF-7 cells. MCF-7 cells were transfected with plasmids expressing Flag-tagged RNF31 or the Flag tag alone. After 48 h, whole-protein extracts were prepared and the levels of RNF31, p53 and the internal control Glyceraldehydes 3-phophate dehydrogenase (GAPDH) were determined by western blot analysis. ( b ) RNF31 depletion increases p53 protein levels. MCF-7 cells were transfected with siRNF31 or siControl. Cells were collected after 24 h. p53 and RNF31 levels were determined by western blot analysis. GAPDH was used as internal control. ( c ) RNF31 depletion does not further increase the stability of p53 in the presence of the proteasome inhibitor MG132. MCF-7 cells were transfected with siRNF31 or siControl. After 48 h, cells were treated with 10 μ M MG132 or vehicle. Cells were collected 6 h after treatment and whole-protein extracts were prepared. The levels of RNF31, p53 and the internal control GAPDH were determined by western blot analysis. ( d ) Depletion of RNF31 increases p53 protein stability. MCF-7 cells were transfected with siRNF31 or siControl. After 48 h, cells were treated with protein biosynthesis inhibitor (100 μ M cycloheximide) for different times before whole-protein extraction. The levels of RNF31, p53 and the internal control GAPDH were determined by western blot analysis. ImageJ was used to quantify the p53 band density, followed by a normalization of the p53 level, with the level at time point zero set as 1. ( e ) Overexpression of RNF31 decreases p53 protein stability. MCF-7 cells were transfected with siRNF31 or siControl. After 48 h, cells were treated with protein biosynthesis inhibitor (100 μ M cycloheximide) for different times before whole-protein extraction. The levels of RNF31, p53 and the internal control GAPDH were determined by western blot analysis. ImageJ was used to quantify the p53 band density, followed by a normalization of the p53 level, with the level at time point zero set as 1.

    Techniques Used: Over Expression, Transfection, Expressing, FLAG-tag, Western Blot, Protein Extraction

    RNF31 associates with p53/MDM2 complex and increases p53 polyubiquitination and degradation in an MDM2-dependent manner. ( a ) Co-immunoprecipitation (Co-IP) assays reveal associations between endogenous RNF31 and the p53/MDM2 complex in MCF-7 cells. IgG was used as control. ( b ) RNF31 associates with the p53/MDM2 complex in HEK293 cells. Cells were seeded in 10-cm dishes. After 24 h, cells were transfected with 2 μg myc-MDM2 plasmid, 2 μg p53 plasmid and 2 μg Flag-RNF31 plasmid. After another 24 h, cells were collected. Co-IP was performed using antibodies as indicated. ( c ) Nutlin-3 does not affect the association between RNF31 and p53. MCF-7 cells were treated with vehicle or Nutlin-3 for 2 h. Co-IP was performed using antibodies as indicated. IgG was used as negative control for IP. IP of MDM2 was used as control for Nutlin-3 function. ImageJ was applied to quantify p53 and MDM2 band densities. ( d ) RNF31 facilitates p53 polyubiquitination. HEK293 cells were transfected with 0.5 μg each of the Flag-RNF31, myc-MDM2 and p53. Green fluorescent protein (GFP) was used as the transfection control. After 24 h, MG132 (10 μ M ) was added. Four hours later, whole-cell extracts were prepared for western blot analysis. ( e ) RNF31 facilitates interaction between p53 and ubiquitin. HEK293 cells were transfected with 0.5 μg each of the Flag-RNF31, myc-MDM2 and p53. After 24 h, MG132 (10 μ M ) was added. Four hours later, whole-cell extracts were prepared and thereafter subjected to immunoprecipitation using ubiquitin antibody. Western blot analysis using p53 antibody was used to detect ubiquitinated p53 forms. The predicted molecular weight of polyubiquitinated p53 is indicated. ( f ) The effect of RNF31 on p53 stability is dependent on interaction between p53 and MDM2. MCF-7 cells were transfected with siRNF31 or siControl and treated with vehicle, 10 μ M cisplatin or 10 μ M nutlin-3 for 24 h. p53 and RNF31 levels were determined by western blot analysis. Glyceraldehydes 3-phophate dehydrogenase (GAPDH) was used as an internal control.
    Figure Legend Snippet: RNF31 associates with p53/MDM2 complex and increases p53 polyubiquitination and degradation in an MDM2-dependent manner. ( a ) Co-immunoprecipitation (Co-IP) assays reveal associations between endogenous RNF31 and the p53/MDM2 complex in MCF-7 cells. IgG was used as control. ( b ) RNF31 associates with the p53/MDM2 complex in HEK293 cells. Cells were seeded in 10-cm dishes. After 24 h, cells were transfected with 2 μg myc-MDM2 plasmid, 2 μg p53 plasmid and 2 μg Flag-RNF31 plasmid. After another 24 h, cells were collected. Co-IP was performed using antibodies as indicated. ( c ) Nutlin-3 does not affect the association between RNF31 and p53. MCF-7 cells were treated with vehicle or Nutlin-3 for 2 h. Co-IP was performed using antibodies as indicated. IgG was used as negative control for IP. IP of MDM2 was used as control for Nutlin-3 function. ImageJ was applied to quantify p53 and MDM2 band densities. ( d ) RNF31 facilitates p53 polyubiquitination. HEK293 cells were transfected with 0.5 μg each of the Flag-RNF31, myc-MDM2 and p53. Green fluorescent protein (GFP) was used as the transfection control. After 24 h, MG132 (10 μ M ) was added. Four hours later, whole-cell extracts were prepared for western blot analysis. ( e ) RNF31 facilitates interaction between p53 and ubiquitin. HEK293 cells were transfected with 0.5 μg each of the Flag-RNF31, myc-MDM2 and p53. After 24 h, MG132 (10 μ M ) was added. Four hours later, whole-cell extracts were prepared and thereafter subjected to immunoprecipitation using ubiquitin antibody. Western blot analysis using p53 antibody was used to detect ubiquitinated p53 forms. The predicted molecular weight of polyubiquitinated p53 is indicated. ( f ) The effect of RNF31 on p53 stability is dependent on interaction between p53 and MDM2. MCF-7 cells were transfected with siRNF31 or siControl and treated with vehicle, 10 μ M cisplatin or 10 μ M nutlin-3 for 24 h. p53 and RNF31 levels were determined by western blot analysis. Glyceraldehydes 3-phophate dehydrogenase (GAPDH) was used as an internal control.

    Techniques Used: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Negative Control, Western Blot, Molecular Weight

    RNF31 depletion induces G1 cell cycle arrest and cisplatin-induced apoptosis in a p53-dependent manner. ( a ) The efficiency of RNF31 and p53 knockdown in MCF-7 cells. RNF31 and p53 protein levels were determined by western blot analysis. Glyceraldehydes 3-phophate dehydrogenase (GAPDH) was used as internal control. A representative blot of three independent experiments is shown. ( b ) RNF31 knockdown decreases cell proliferation in MCF-7 cells as determined by EdU incorporation, which can be rescued by p53 depletion. MCF-7 cells were treated with siControl, siRNF31 or siRNF31 and siP53 for 48 h. EdU was added at a concentration of 10 μ M and incubated for 1 h. The cells were subject to fluorescence-activated cell sorting (FACS) analysis. All values are mean±s.d. ( n =3, * P
    Figure Legend Snippet: RNF31 depletion induces G1 cell cycle arrest and cisplatin-induced apoptosis in a p53-dependent manner. ( a ) The efficiency of RNF31 and p53 knockdown in MCF-7 cells. RNF31 and p53 protein levels were determined by western blot analysis. Glyceraldehydes 3-phophate dehydrogenase (GAPDH) was used as internal control. A representative blot of three independent experiments is shown. ( b ) RNF31 knockdown decreases cell proliferation in MCF-7 cells as determined by EdU incorporation, which can be rescued by p53 depletion. MCF-7 cells were treated with siControl, siRNF31 or siRNF31 and siP53 for 48 h. EdU was added at a concentration of 10 μ M and incubated for 1 h. The cells were subject to fluorescence-activated cell sorting (FACS) analysis. All values are mean±s.d. ( n =3, * P

    Techniques Used: Western Blot, Concentration Assay, Incubation, Fluorescence, FACS

    RNF31 depletion increases p53 protein levels and expression of p53 target genes. ( a ) RNF31 depletion increases p53 protein levels using four different siRNA oligos. MCF-7 cells were transfected with siRNF31 or siControl. After 72 h, p53 and RNF31 levels were determined by western blot analysis. Glyceraldehydes 3-phophate dehydrogenase (GAPDH) was used as internal control. ( b ) RNF31 depletion increases p53 target genes using four different siRNA oligos. MCF-7 cells were transfected with siRNF31 or siControl. After 72 h RNA was prepared and the expression of the endogenous p53 target genes, P21 , IGFBP3 and BTG2 , were determined by quantitative PCR (qPCR). Shown are the results from three experiments. n =3, * P
    Figure Legend Snippet: RNF31 depletion increases p53 protein levels and expression of p53 target genes. ( a ) RNF31 depletion increases p53 protein levels using four different siRNA oligos. MCF-7 cells were transfected with siRNF31 or siControl. After 72 h, p53 and RNF31 levels were determined by western blot analysis. Glyceraldehydes 3-phophate dehydrogenase (GAPDH) was used as internal control. ( b ) RNF31 depletion increases p53 target genes using four different siRNA oligos. MCF-7 cells were transfected with siRNF31 or siControl. After 72 h RNA was prepared and the expression of the endogenous p53 target genes, P21 , IGFBP3 and BTG2 , were determined by quantitative PCR (qPCR). Shown are the results from three experiments. n =3, * P

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

    25) Product Images from "Activation of KLF1 Enhances the Differentiation and Maturation of Red Blood Cells from Human Pluripotent Stem Cells"

    Article Title: Activation of KLF1 Enhances the Differentiation and Maturation of Red Blood Cells from Human Pluripotent Stem Cells

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.2562

    Functional assessment of KLF1‐ER T2 and R328L‐ER T2 fusion proteins. ( A, B) : Western blot analyses of cell lysates isolated from untransfected COS7 cells (lane 1), COS7 cells transfected with pCAG‐KLF1 (lane 3); pCAG‐R328L (lane 4); pCAG‐KLF1‐ER T2 (lane 5); pCAG‐R328L‐ER T2 (lane 6) and the murine CAG‐ KLF1‐ER T2 (lane 7) using an anti‐KLF1 antibody (A) and GAPDH as a loading control (B). Lane 2 is blank. ( C, D) : Immunofluorescence staining of COS7 cells transfected with either the CAG‐KLF1‐ER T2 (A) or CAG‐R328L‐ER T2 (B) constructs then stained with anti‐KLF1 antibodies (green) and the DAPI nuclear dye (blue) in the presence and absence of tamoxifen as indicated. (Scale bar 10 μm). (E) : Quantitative reverse‐transcriptase polymerase chain reaction analyses of RNA isolated from control and hemin and/or tamoxifen‐treated K562 cells and K562 cells transfected with either CAG‐KLF1‐ER T2 or CAG‐R328L‐ER T2 vectors using primers for the KLF1 target gene, AHSP . Data represent three independent experiments and error bars represent SEM. p values were calculated using using one‐way ANOVA followed Tukey's multiple comparisons test. (* p
    Figure Legend Snippet: Functional assessment of KLF1‐ER T2 and R328L‐ER T2 fusion proteins. ( A, B) : Western blot analyses of cell lysates isolated from untransfected COS7 cells (lane 1), COS7 cells transfected with pCAG‐KLF1 (lane 3); pCAG‐R328L (lane 4); pCAG‐KLF1‐ER T2 (lane 5); pCAG‐R328L‐ER T2 (lane 6) and the murine CAG‐ KLF1‐ER T2 (lane 7) using an anti‐KLF1 antibody (A) and GAPDH as a loading control (B). Lane 2 is blank. ( C, D) : Immunofluorescence staining of COS7 cells transfected with either the CAG‐KLF1‐ER T2 (A) or CAG‐R328L‐ER T2 (B) constructs then stained with anti‐KLF1 antibodies (green) and the DAPI nuclear dye (blue) in the presence and absence of tamoxifen as indicated. (Scale bar 10 μm). (E) : Quantitative reverse‐transcriptase polymerase chain reaction analyses of RNA isolated from control and hemin and/or tamoxifen‐treated K562 cells and K562 cells transfected with either CAG‐KLF1‐ER T2 or CAG‐R328L‐ER T2 vectors using primers for the KLF1 target gene, AHSP . Data represent three independent experiments and error bars represent SEM. p values were calculated using using one‐way ANOVA followed Tukey's multiple comparisons test. (* p

    Techniques Used: Functional Assay, Western Blot, Isolation, Transfection, Immunofluorescence, Staining, Construct, Polymerase Chain Reaction

    26) Product Images from "miR-4317 suppresses non-small cell lung cancer (NSCLC) by targeting fibroblast growth factor 9 (FGF9) and cyclin D2 (CCND2)"

    Article Title: miR-4317 suppresses non-small cell lung cancer (NSCLC) by targeting fibroblast growth factor 9 (FGF9) and cyclin D2 (CCND2)

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0882-4

    FGF9 and CCND2 were two direct target genes of miR-4317. a , b TGFBR1 and CCNE2 were identified as potential regulatory targets of miR-4317 by considering the downregulation of genes using prediction tools and qRT-PCR. c , d The expression levels of FGF9 and CCND2 mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection of miR-4317 mimic in the A973 and A549 cell lines, respectively. e , f The expression levels of FGF9 and CCND2 mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection of miR-4317 inhibitors in the GLC82 and H157 cell lines, respectively. g , h Dual-luciferase reporter assay. The relative luciferase activity was normalized to the Renilla luciferase activity assay after co-transfection of cells with miR-4317 mimic and pmiR-RB-REPORT™ constructs containing WT or MUT FGF9 and the CCND2 3’-UTR region in A973 and A549 cell lines. Data are presented as the mean values ± SD from triplicate experiments. ♦ p
    Figure Legend Snippet: FGF9 and CCND2 were two direct target genes of miR-4317. a , b TGFBR1 and CCNE2 were identified as potential regulatory targets of miR-4317 by considering the downregulation of genes using prediction tools and qRT-PCR. c , d The expression levels of FGF9 and CCND2 mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection of miR-4317 mimic in the A973 and A549 cell lines, respectively. e , f The expression levels of FGF9 and CCND2 mRNA and protein were measured by qRT-PCR and western blot analysis using GAPDH as the loading control after transfection of miR-4317 inhibitors in the GLC82 and H157 cell lines, respectively. g , h Dual-luciferase reporter assay. The relative luciferase activity was normalized to the Renilla luciferase activity assay after co-transfection of cells with miR-4317 mimic and pmiR-RB-REPORT™ constructs containing WT or MUT FGF9 and the CCND2 3’-UTR region in A973 and A549 cell lines. Data are presented as the mean values ± SD from triplicate experiments. ♦ p

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Activity Assay, Cotransfection, Construct

    27) Product Images from "Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor"

    Article Title: Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1824

    MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.
    Figure Legend Snippet: MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, shRNA, Fractionation, SDS Page, Purification, Incubation, Immunofluorescence

    MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF RNA levels as determined by qRT-PCR. Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.
    Figure Legend Snippet: MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF RNA levels as determined by qRT-PCR. Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.

    Techniques Used: Infection, shRNA, Quantitative RT-PCR, Expressing

    28) Product Images from "Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells"

    Article Title: Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddq520

    Human ESCs (H9 and HSF1) and iPSCs [iPS(IMR90) and iHUF4] were differentiated as adherent cultures with 20% fetal bovine serum medium supplemented with BMP-4, -7, and -8b. ( A ) Morphology of undifferentiated and differentiated cells; after 14 days of differentiation, cultures appeared confluent, with cell morphology distinct from undifferentiated cells for all cell lines. ( B ) Western blot analysis of germ cell-specific proteins VASA and DAZL in undifferentiated cells and cells differentiated for 7 and 14 days. Increased expression of both VASA and DAZL was observed for all cell lines with differentiation, with the level of expression being similar at day 7 and day 14 time points between all the cell lines. Notably, a low level of VASA and DAZL protein expression was detected for undifferentiated iHUF4 cells and VASA expression in one sample of undifferentiated iPS(IMR90) cells. GAPDH is shown as a loading control and 293T cells are used as a negative control. Two independent samples are shown for each time point for all cell lines. ( C ) The expression and cytoplasmic localization of the VASA protein was also detected by immunostaining for all cell lines after differentiation. In addition, occasional cells in undifferentiated cultures were stained positive for all cell lines. Representative images are shown for iHUF4 and iPS(IMR90) cells; green VASA, blue DAPI, merged image and lower magnification image showing the surrounding cells. Scale bars: 200 µm (A); 50 µm (C).
    Figure Legend Snippet: Human ESCs (H9 and HSF1) and iPSCs [iPS(IMR90) and iHUF4] were differentiated as adherent cultures with 20% fetal bovine serum medium supplemented with BMP-4, -7, and -8b. ( A ) Morphology of undifferentiated and differentiated cells; after 14 days of differentiation, cultures appeared confluent, with cell morphology distinct from undifferentiated cells for all cell lines. ( B ) Western blot analysis of germ cell-specific proteins VASA and DAZL in undifferentiated cells and cells differentiated for 7 and 14 days. Increased expression of both VASA and DAZL was observed for all cell lines with differentiation, with the level of expression being similar at day 7 and day 14 time points between all the cell lines. Notably, a low level of VASA and DAZL protein expression was detected for undifferentiated iHUF4 cells and VASA expression in one sample of undifferentiated iPS(IMR90) cells. GAPDH is shown as a loading control and 293T cells are used as a negative control. Two independent samples are shown for each time point for all cell lines. ( C ) The expression and cytoplasmic localization of the VASA protein was also detected by immunostaining for all cell lines after differentiation. In addition, occasional cells in undifferentiated cultures were stained positive for all cell lines. Representative images are shown for iHUF4 and iPS(IMR90) cells; green VASA, blue DAPI, merged image and lower magnification image showing the surrounding cells. Scale bars: 200 µm (A); 50 µm (C).

    Techniques Used: Western Blot, Expressing, Negative Control, Immunostaining, Staining

    29) Product Images from "Circular RNA circ-Foxo3 induced cell apoptosis in urothelial carcinoma via interaction with miR-191-5p"

    Article Title: Circular RNA circ-Foxo3 induced cell apoptosis in urothelial carcinoma via interaction with miR-191-5p

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S215823

    circ-Foxo3 induced cell apoptosis and reduced viability in bladder cancer cells. T24, UM-UC-3, and J82 bladder cancer cell lines received control (Ctrl) or circ-Foxo3 over-expression ( circ-Foxo3 ) plasmids. ( A ) Expression of circ-Foxo3 RNA level in bladder cell lines. ( B ) Western blot of cleaved-caspase3 (c-caspase3), Bax, Bcl2 and GAPDH expression in cells receiving treatments. The band intensity relative to GAPDH, and the ratio of Bax/Bcl2 band intensity were quantified (n=5). ( C ) Analysis of apoptosis in treated bladder cancer cell lines by flow cytometry (n=5). ( D ) Cell viability measured by the CCK-8 assay in T24, UM-UC-3, and J82 bladder cancer cell lines. Mean ± SD, * p
    Figure Legend Snippet: circ-Foxo3 induced cell apoptosis and reduced viability in bladder cancer cells. T24, UM-UC-3, and J82 bladder cancer cell lines received control (Ctrl) or circ-Foxo3 over-expression ( circ-Foxo3 ) plasmids. ( A ) Expression of circ-Foxo3 RNA level in bladder cell lines. ( B ) Western blot of cleaved-caspase3 (c-caspase3), Bax, Bcl2 and GAPDH expression in cells receiving treatments. The band intensity relative to GAPDH, and the ratio of Bax/Bcl2 band intensity were quantified (n=5). ( C ) Analysis of apoptosis in treated bladder cancer cell lines by flow cytometry (n=5). ( D ) Cell viability measured by the CCK-8 assay in T24, UM-UC-3, and J82 bladder cancer cell lines. Mean ± SD, * p

    Techniques Used: Over Expression, Expressing, Western Blot, Flow Cytometry, Cytometry, CCK-8 Assay

    circ-Foxo3 promoted bladder tumor cell apoptosis in BBN mice. Male, 8-week-old C57BL/6 mice received control (circControl-GFP) and circ-Foxo3 (circFoxo3-GFP) somatic gene transfer by lentiviral injection, followed by exposure to oral vehicle or BBN treatment for 17 weeks. ( A ) The bladder tumor cell apoptosis was evaluated by the TUNEL assay (left) and quantified (right; n=10). ( B )Western blot of cleaved-caspase3 (c-caspase3), Bax, Bcl2 and GAPDH expression in murine bladder tissues. The band intensity relative to GAPDH, and the ratio of Bax/Bcl2 band intensity were quantified (n=5). Mean ± SD, * p
    Figure Legend Snippet: circ-Foxo3 promoted bladder tumor cell apoptosis in BBN mice. Male, 8-week-old C57BL/6 mice received control (circControl-GFP) and circ-Foxo3 (circFoxo3-GFP) somatic gene transfer by lentiviral injection, followed by exposure to oral vehicle or BBN treatment for 17 weeks. ( A ) The bladder tumor cell apoptosis was evaluated by the TUNEL assay (left) and quantified (right; n=10). ( B )Western blot of cleaved-caspase3 (c-caspase3), Bax, Bcl2 and GAPDH expression in murine bladder tissues. The band intensity relative to GAPDH, and the ratio of Bax/Bcl2 band intensity were quantified (n=5). Mean ± SD, * p

    Techniques Used: Mouse Assay, Injection, TUNEL Assay, Western Blot, Expressing

    circ-Foxo3 was upregulated in response to apoptotic stress. ( A ) Western blot of cleaved-caspase3 (c-caspase3), Bax, Bcl2 and GAPDH expression in J82 cells treated with vehicle (Ctrl), doxorubicin (Dox; 1μg/mL), cisplatin (CP; 2 μg/mL), and H 2 O 2 (H2O2; 0.4 mM). The band intensity relative to GAPDH, and the ratio of Bax/Bcl2 band intensity were shown on the right. ( B – D ) Expression of circ-Foxo3 RNA level in bladder cell lines treated with vehicle ( B – D ), doxorubicin ( B ), cisplatin ( C ), and H 2 O 2 ( D ), respectively (n=5). Mean ± SD, * p
    Figure Legend Snippet: circ-Foxo3 was upregulated in response to apoptotic stress. ( A ) Western blot of cleaved-caspase3 (c-caspase3), Bax, Bcl2 and GAPDH expression in J82 cells treated with vehicle (Ctrl), doxorubicin (Dox; 1μg/mL), cisplatin (CP; 2 μg/mL), and H 2 O 2 (H2O2; 0.4 mM). The band intensity relative to GAPDH, and the ratio of Bax/Bcl2 band intensity were shown on the right. ( B – D ) Expression of circ-Foxo3 RNA level in bladder cell lines treated with vehicle ( B – D ), doxorubicin ( B ), cisplatin ( C ), and H 2 O 2 ( D ), respectively (n=5). Mean ± SD, * p

    Techniques Used: Western Blot, Expressing

    miR-191-5p suppressed the pro-apoptotic effect of circ-Foxo3 in bladder cancer cells. ( A ) T24, UM-UC-3, and J82 bladder cancer cell lines were transfected with miR-191 mimic (10 nM) and treated with cisplatin (CP, 2 μg/mL). The expression of cleaved-caspase3 (c-caspase3) was examined by Western blot and quantified relative to GAPDH (n=5). ( B – C ) T24, UM-UC-3, and J82 bladder cancer cell lines were co-transfected with miR-191 mimic and a circ-Foxo3 over-expression plasmid (n=5). The expression of cleaved-caspase3 (c-caspase3) was examined by Western blot (B) and apoptosis was analyzed by flow cytometry ( C ). ( D ) Cell viability measured by the CCK-8 assay in T24, UM-UC-3, and J82 bladder cancer cell lines receiving miR-191 mimic or circ-Foxo3 transfection (n=5). Mean ± SD, * p
    Figure Legend Snippet: miR-191-5p suppressed the pro-apoptotic effect of circ-Foxo3 in bladder cancer cells. ( A ) T24, UM-UC-3, and J82 bladder cancer cell lines were transfected with miR-191 mimic (10 nM) and treated with cisplatin (CP, 2 μg/mL). The expression of cleaved-caspase3 (c-caspase3) was examined by Western blot and quantified relative to GAPDH (n=5). ( B – C ) T24, UM-UC-3, and J82 bladder cancer cell lines were co-transfected with miR-191 mimic and a circ-Foxo3 over-expression plasmid (n=5). The expression of cleaved-caspase3 (c-caspase3) was examined by Western blot (B) and apoptosis was analyzed by flow cytometry ( C ). ( D ) Cell viability measured by the CCK-8 assay in T24, UM-UC-3, and J82 bladder cancer cell lines receiving miR-191 mimic or circ-Foxo3 transfection (n=5). Mean ± SD, * p

    Techniques Used: Transfection, Expressing, Western Blot, Over Expression, Plasmid Preparation, Flow Cytometry, Cytometry, CCK-8 Assay

    30) Product Images from "Cooperativity and Equilibrium with FOXA1 Define the Androgen Receptor Transcriptional Program"

    Article Title: Cooperativity and Equilibrium with FOXA1 Define the Androgen Receptor Transcriptional Program

    Journal: Nature communications

    doi: 10.1038/ncomms4972

    FOXA1 downregulation results in AR-chromatin binding in the absence of androgen (A) QRT-PCR and immunoblot analyses of AR, FOXA1 and KLK3 were performed in hormone-starved control and shFOXA1 LNCaP cells. GAPDH was used as a loading control. Error bars indicate n=3, mean±s.e.m., p
    Figure Legend Snippet: FOXA1 downregulation results in AR-chromatin binding in the absence of androgen (A) QRT-PCR and immunoblot analyses of AR, FOXA1 and KLK3 were performed in hormone-starved control and shFOXA1 LNCaP cells. GAPDH was used as a loading control. Error bars indicate n=3, mean±s.e.m., p

    Techniques Used: Binding Assay, Quantitative RT-PCR

    31) Product Images from "β-Arrestin1/miR-326 Transcription Unit Is Epigenetically Regulated in Neural Stem Cells Where It Controls Stemness and Growth Arrest"

    Article Title: β-Arrestin1/miR-326 Transcription Unit Is Epigenetically Regulated in Neural Stem Cells Where It Controls Stemness and Growth Arrest

    Journal: Stem Cells International

    doi: 10.1155/2017/5274171

    β arr-1 overexpression impairs NSCs clonogenicity and proliferation. (a) Representative image of immunofluorescence staining of NSCs after overexpression of HA- β arr-1. Nuclei are counterstained with Hoechst. Scale bar: 5 μ m. (b) WB analysis of endogenous stemness markers in NSCs after overexpression of HA- β arr-1. Loading control: actin. Representative Western blot images from 3 independent experiments. (c) mRNA expression levels of stemness markers in NSCs after overexpression of HA- β arr-1. (d) Oncosphere forming assay (number of colonies, left panel) and bromodeoxyuridine (BrdU) uptake (right panel) in NSCs after ectopic expression of HA- β arr-1. (e) Cell viability (MTT assay) in NSC after ectopic expression of HA- β arr-1. (f) WB analysis of cleaved Parp (Parp-c) in NSCs after overexpression of HA- β arr-1. Loading control: Gapdh. Representative Western blot images from 3 independent experiments. ((c), (d), and (e)) Data are means ± SD from 3 independent experiments. ∗ p
    Figure Legend Snippet: β arr-1 overexpression impairs NSCs clonogenicity and proliferation. (a) Representative image of immunofluorescence staining of NSCs after overexpression of HA- β arr-1. Nuclei are counterstained with Hoechst. Scale bar: 5 μ m. (b) WB analysis of endogenous stemness markers in NSCs after overexpression of HA- β arr-1. Loading control: actin. Representative Western blot images from 3 independent experiments. (c) mRNA expression levels of stemness markers in NSCs after overexpression of HA- β arr-1. (d) Oncosphere forming assay (number of colonies, left panel) and bromodeoxyuridine (BrdU) uptake (right panel) in NSCs after ectopic expression of HA- β arr-1. (e) Cell viability (MTT assay) in NSC after ectopic expression of HA- β arr-1. (f) WB analysis of cleaved Parp (Parp-c) in NSCs after overexpression of HA- β arr-1. Loading control: Gapdh. Representative Western blot images from 3 independent experiments. ((c), (d), and (e)) Data are means ± SD from 3 independent experiments. ∗ p

    Techniques Used: Over Expression, Immunofluorescence, Staining, Western Blot, Expressing, MTT Assay

    β arr-1 controls NSCs proliferation via p27 expression. (a) β arr-1 and p27 mRNA expression levels in NSCs transfected with the HA- β arr-1 plasmid for 48 hrs. (b) Subcellular localization of β arr-1 and p27 proteins in NSCs transfected with the HA- β arr-1 plasmid and analyzed 48 hrs after transfection. Proteins are shown in the cytosolic (Cyto) and nuclear (Nuc) fractions. Sp1: nuclear loading control; Gapdh: cytoplasmic loading control. Representative Western blot images from 3 independent experiments. (c) βarr-1 and p27 mRNA expression levels in NSCs transfected with control siRNA (s iCtr) or β arr-1 siRNA (si β arr-1) cultured for 24 hrs in DFM. (d) Western blot showing endogenous β arr-1 and p27 protein levels in NSCs transfected with control siRNA (siCtr) or β arr-1 siRNA (si β arr-1) cultured for 24 hrs in DFM. Representative Western blot images from 3 independent experiments. ((a) and (c)) Data are means ± SD from 3 independent experiments. ∗ p
    Figure Legend Snippet: β arr-1 controls NSCs proliferation via p27 expression. (a) β arr-1 and p27 mRNA expression levels in NSCs transfected with the HA- β arr-1 plasmid for 48 hrs. (b) Subcellular localization of β arr-1 and p27 proteins in NSCs transfected with the HA- β arr-1 plasmid and analyzed 48 hrs after transfection. Proteins are shown in the cytosolic (Cyto) and nuclear (Nuc) fractions. Sp1: nuclear loading control; Gapdh: cytoplasmic loading control. Representative Western blot images from 3 independent experiments. (c) βarr-1 and p27 mRNA expression levels in NSCs transfected with control siRNA (s iCtr) or β arr-1 siRNA (si β arr-1) cultured for 24 hrs in DFM. (d) Western blot showing endogenous β arr-1 and p27 protein levels in NSCs transfected with control siRNA (siCtr) or β arr-1 siRNA (si β arr-1) cultured for 24 hrs in DFM. Representative Western blot images from 3 independent experiments. ((a) and (c)) Data are means ± SD from 3 independent experiments. ∗ p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Cell Culture

    32) Product Images from "Suitability of Yin Yang 1 transcript and protein levels for biomarker studies in B cell non-Hodgkin lymphoma"

    Article Title: Suitability of Yin Yang 1 transcript and protein levels for biomarker studies in B cell non-Hodgkin lymphoma

    Journal: Biomarker Research

    doi: 10.1186/s40364-018-0126-y

    Cellular stability of YY1 mRNA and protein. Influence of the transcriptional inhibitor actinomycin-D (ACT-D, 5 μg/mL) ( a - d ) and of the translational inhibitor cycloheximide (CHX, 100 μg/mL) ( e , f ) on YY1 expression in the B-NHL cell lines RAMOS ( a , b and e ) and U2932-R2 ( c , d and f ). a YY1 mRNA quantification relative to GAPDH by qRT-PCR after 0 h, 30 min, 1 h, 2 h, 3 h and 6 h of ACT-D treatment of RAMOS and ( c ) of U2932-R2. Error bars indicate 95% confidence interval of the mean expression. MYC levels were analyzed as a positive control for a fast turnover mRNA. b Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h of ACT-D treatment in RAMOS and ( d ) in U2932-R2. MYC was used as a positive control for a fast turnover protein and GAPDH as loading control. The arrow indicates the caspase-cleaved YY1 form. e Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h CHX treatment of RAMOS and ( f ) of U2932-R2. MYC expression was determined as a positive control for a fast turnover protein and GAPDH and histone 3 (H3) served as loading controls. The arrow indicates the caspase-cleaved YY1 form. Numbers underneath the blots refer to the relative amount of YY1 normalized to GAPDH levels according to densitometric analyses of the blots. The 0 h sample was set to 1 at each time point, treatment and cell line
    Figure Legend Snippet: Cellular stability of YY1 mRNA and protein. Influence of the transcriptional inhibitor actinomycin-D (ACT-D, 5 μg/mL) ( a - d ) and of the translational inhibitor cycloheximide (CHX, 100 μg/mL) ( e , f ) on YY1 expression in the B-NHL cell lines RAMOS ( a , b and e ) and U2932-R2 ( c , d and f ). a YY1 mRNA quantification relative to GAPDH by qRT-PCR after 0 h, 30 min, 1 h, 2 h, 3 h and 6 h of ACT-D treatment of RAMOS and ( c ) of U2932-R2. Error bars indicate 95% confidence interval of the mean expression. MYC levels were analyzed as a positive control for a fast turnover mRNA. b Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h of ACT-D treatment in RAMOS and ( d ) in U2932-R2. MYC was used as a positive control for a fast turnover protein and GAPDH as loading control. The arrow indicates the caspase-cleaved YY1 form. e Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h CHX treatment of RAMOS and ( f ) of U2932-R2. MYC expression was determined as a positive control for a fast turnover protein and GAPDH and histone 3 (H3) served as loading controls. The arrow indicates the caspase-cleaved YY1 form. Numbers underneath the blots refer to the relative amount of YY1 normalized to GAPDH levels according to densitometric analyses of the blots. The 0 h sample was set to 1 at each time point, treatment and cell line

    Techniques Used: Activated Clotting Time Assay, Expressing, Quantitative RT-PCR, Positive Control, Western Blot

    33) Product Images from "Role of Nox2 and p22phox in Persistent Postoperative Hypertension in Aldosterone-Producing Adenoma Patients after Adrenalectomy"

    Article Title: Role of Nox2 and p22phox in Persistent Postoperative Hypertension in Aldosterone-Producing Adenoma Patients after Adrenalectomy

    Journal: International Journal of Endocrinology

    doi: 10.1155/2016/2395634

    Activity and expression of Nox in different adrenal tissues. (a) Nox activity was detected in normal adrenocortical tissue, APA, and NFA by lucigenin chemiluminescence. (b) Expression of Nox1 – Nox5 , Duox1 , Duox2 , and p22phox mRNA in normal adrenocortical tissue and APA by RT-PCR. Signals for Nox1 , Nox2 , Nox4 , Duox1 , and p22phox were detected in normal adrenal tissue and APA. (c–h) Relative expression of Nox1 , Nox2 , Nox4 , Duox1 , p22phox , and CYP11B2 in normal adrenocortical tissue, APA, and NFA measured by Q-PCR. Nox2 , p22phox , and CYP11B2 were primarily expressed in APA. (i-j) Correlation between CYP11B2 mRNA with Nox2 , CYP11B2 , and p22phox mRNA in APA. A positive correlation between Nox2 and CYP11B2 mRNA was found. (k–n) Western blot analysis of Nox2 and p22phox in normal adrenocortical tissue, APA, and NFA. Nox2 and p22phox were enhanced in APA compared with normal adrenocortical tissue and NFA. Results of densitometric analysis of Nox2 and p22phox proteins normalized to GAPDH were shown.
    Figure Legend Snippet: Activity and expression of Nox in different adrenal tissues. (a) Nox activity was detected in normal adrenocortical tissue, APA, and NFA by lucigenin chemiluminescence. (b) Expression of Nox1 – Nox5 , Duox1 , Duox2 , and p22phox mRNA in normal adrenocortical tissue and APA by RT-PCR. Signals for Nox1 , Nox2 , Nox4 , Duox1 , and p22phox were detected in normal adrenal tissue and APA. (c–h) Relative expression of Nox1 , Nox2 , Nox4 , Duox1 , p22phox , and CYP11B2 in normal adrenocortical tissue, APA, and NFA measured by Q-PCR. Nox2 , p22phox , and CYP11B2 were primarily expressed in APA. (i-j) Correlation between CYP11B2 mRNA with Nox2 , CYP11B2 , and p22phox mRNA in APA. A positive correlation between Nox2 and CYP11B2 mRNA was found. (k–n) Western blot analysis of Nox2 and p22phox in normal adrenocortical tissue, APA, and NFA. Nox2 and p22phox were enhanced in APA compared with normal adrenocortical tissue and NFA. Results of densitometric analysis of Nox2 and p22phox proteins normalized to GAPDH were shown.

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

    34) Product Images from "Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing"

    Article Title: Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr076

    Nuclear localization of Dicer and Ago2. ( A ) Western analysis of Dicer in cytoplasmic and nuclear fractions. The 5% cytoplasmic and 20% nuclear fractions prepared from HeLa cells were loaded in a 4–12% SDS–PAGE gel, transferred to a membrane and proteins were detected using antibodies. Alpha-tubulin and hnRNP A2 were used as controls for cytoplasmic and nuclear proteins, respectively. ( B ) Ago2 can be found in both cytoplasmic and nuclear fractions. Western analysis was performed using the same samples as in ( C ), and the membrane was probed using different antibodies. GAPDH and hnRNPA2 were detected and used as controls for cytoplasmic and nuclear proteins, respectively. (C) Localization of Dicer (upper panel) and Ago2 (lower panel) in Hela cells. Cells grown on glass-bottom dishes were fixed, stained with first antibodies against Dicer (1:200, ab14601, from mouse, Abcam) and Nucleolin (1:200, ab22758, from rabbit, Abcam), or against Ago2 (1:150, ab57113, from mouse, Abcam) and Nucleolin, as described in ‘Materials and Methods’ section. Secondary antibodies were anti-mouse antibody (1:200, ab6785, Abcam, conjugated with FITC (green) and anti-rabbit antibody (1:200, Ab6719, conjugated with Texas Red). Nucleolin (red) was detected and served as a nucleolar marker. The nucleus was stained with DAPI (blue). The arrow indicates the positions of nucleoli. The scale bars: 10 µm.
    Figure Legend Snippet: Nuclear localization of Dicer and Ago2. ( A ) Western analysis of Dicer in cytoplasmic and nuclear fractions. The 5% cytoplasmic and 20% nuclear fractions prepared from HeLa cells were loaded in a 4–12% SDS–PAGE gel, transferred to a membrane and proteins were detected using antibodies. Alpha-tubulin and hnRNP A2 were used as controls for cytoplasmic and nuclear proteins, respectively. ( B ) Ago2 can be found in both cytoplasmic and nuclear fractions. Western analysis was performed using the same samples as in ( C ), and the membrane was probed using different antibodies. GAPDH and hnRNPA2 were detected and used as controls for cytoplasmic and nuclear proteins, respectively. (C) Localization of Dicer (upper panel) and Ago2 (lower panel) in Hela cells. Cells grown on glass-bottom dishes were fixed, stained with first antibodies against Dicer (1:200, ab14601, from mouse, Abcam) and Nucleolin (1:200, ab22758, from rabbit, Abcam), or against Ago2 (1:150, ab57113, from mouse, Abcam) and Nucleolin, as described in ‘Materials and Methods’ section. Secondary antibodies were anti-mouse antibody (1:200, ab6785, Abcam, conjugated with FITC (green) and anti-rabbit antibody (1:200, Ab6719, conjugated with Texas Red). Nucleolin (red) was detected and served as a nucleolar marker. The nucleus was stained with DAPI (blue). The arrow indicates the positions of nucleoli. The scale bars: 10 µm.

    Techniques Used: Western Blot, SDS Page, Staining, Marker

    35) Product Images from "Achyranthes bidentata Polypeptide Protects Schwann Cells From Apoptosis in Hydrogen Peroxide-Induced Oxidative Stress"

    Article Title: Achyranthes bidentata Polypeptide Protects Schwann Cells From Apoptosis in Hydrogen Peroxide-Induced Oxidative Stress

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00868

    ABPPk treatment activates the PI3K/AKT and ERK1/2 pathways in SCs. (A) SCs were treated with 400 μM H 2 O 2 with or without 0.5 μg/ml ABPPk. The cells were collected and analyzed by Western blot for the expression of p-PI3K and PI3K, p-AKT and AKT, and p-ERK1/2 and ERK1/2. (B) Histogram shows data of p-PI3K/PI3K, p-AKT/AKT, and p-ERK/ERK levels from Western blot analyses. GAPDH was used as the protein loading control and band density normalization. ABPPk vs. control: P
    Figure Legend Snippet: ABPPk treatment activates the PI3K/AKT and ERK1/2 pathways in SCs. (A) SCs were treated with 400 μM H 2 O 2 with or without 0.5 μg/ml ABPPk. The cells were collected and analyzed by Western blot for the expression of p-PI3K and PI3K, p-AKT and AKT, and p-ERK1/2 and ERK1/2. (B) Histogram shows data of p-PI3K/PI3K, p-AKT/AKT, and p-ERK/ERK levels from Western blot analyses. GAPDH was used as the protein loading control and band density normalization. ABPPk vs. control: P

    Techniques Used: Western Blot, Expressing

    36) Product Images from "Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment"

    Article Title: Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2018.03.140

    Btk is required for cytokine production in TLR4 and TLR7/8 signalling . M-CSF-differentiated macrophages were (A, B) serum starved for 2h prior to stimulation with 10 ng/ml LPS or 1 μg/ml R848 up to 20 min. Btk was immunoprecipitated using anti-Btk antibody followed by western blotting with 4G10 antibody. Anti-rabbit IgG as isotope control. (C) Macrophages infected with adenovirus at moi 10 to 150 were left for 48 h. Btk expression in cell lysates by Western blot using GAPDH as a protein loading control. (D, E) Following infection, cells were stimulated with either LPS (10 ng/ml) or R848 (1 μg/ml) for 18h and TNF production assessed by ELISA. Btk over-expression was normalised to control (black bar; empty adenovirus) for each moi. (F) Monocytes transfected with Btk siRNA (siBtk#9 and #11) or control siRNA (siCon) at 200 nM then M-CSF-treated for 4 days and Btk expression assessed by Western blot. (G, H) TNF or IL-6 in supernatants was measured by ELISA from siRNA manipulated cells after 18hr with either no stimulus or 1ug/ml R848. (I) Monocytes were transfected with Btk siRNA (siBtk) or control siRNA (siCon) at 100, 200 or 300 nM and then M-CSF-treated for 4 days. B- was no transfection; B+ transfection with buffer alone. Cell lysate expression of Btk was assessed by Western blot. (J) Monocytes were incubated with no oligo (nil), control oligo or BTK oligo at 200 nM prior to nucleofection. After 24 h of M-CSF treatment, media was replaced, and after a further 24 h cells were plated and stimulated ± IFN (1 ng/ml) for 5 min. Cell lysates were used to determine the phosphorylation status of STAT1 and levels of BTK by SDS-PAGE and western blotting. (K, L) Monocytes were transfected with 200 nM siBtk or siCon, M-CSF-treated for 4 days, and then stimulated with either LPS (10 ng/ml) or R848 (1 μg/ml) for 18h; TNF concentration was assessed by ELISA. Cytokine levels are expressed as means of triplicate repeats ± SEM. Results shown combined data from 3 to 6 separate donors. Statistical analysis: student's t-test, ***p
    Figure Legend Snippet: Btk is required for cytokine production in TLR4 and TLR7/8 signalling . M-CSF-differentiated macrophages were (A, B) serum starved for 2h prior to stimulation with 10 ng/ml LPS or 1 μg/ml R848 up to 20 min. Btk was immunoprecipitated using anti-Btk antibody followed by western blotting with 4G10 antibody. Anti-rabbit IgG as isotope control. (C) Macrophages infected with adenovirus at moi 10 to 150 were left for 48 h. Btk expression in cell lysates by Western blot using GAPDH as a protein loading control. (D, E) Following infection, cells were stimulated with either LPS (10 ng/ml) or R848 (1 μg/ml) for 18h and TNF production assessed by ELISA. Btk over-expression was normalised to control (black bar; empty adenovirus) for each moi. (F) Monocytes transfected with Btk siRNA (siBtk#9 and #11) or control siRNA (siCon) at 200 nM then M-CSF-treated for 4 days and Btk expression assessed by Western blot. (G, H) TNF or IL-6 in supernatants was measured by ELISA from siRNA manipulated cells after 18hr with either no stimulus or 1ug/ml R848. (I) Monocytes were transfected with Btk siRNA (siBtk) or control siRNA (siCon) at 100, 200 or 300 nM and then M-CSF-treated for 4 days. B- was no transfection; B+ transfection with buffer alone. Cell lysate expression of Btk was assessed by Western blot. (J) Monocytes were incubated with no oligo (nil), control oligo or BTK oligo at 200 nM prior to nucleofection. After 24 h of M-CSF treatment, media was replaced, and after a further 24 h cells were plated and stimulated ± IFN (1 ng/ml) for 5 min. Cell lysates were used to determine the phosphorylation status of STAT1 and levels of BTK by SDS-PAGE and western blotting. (K, L) Monocytes were transfected with 200 nM siBtk or siCon, M-CSF-treated for 4 days, and then stimulated with either LPS (10 ng/ml) or R848 (1 μg/ml) for 18h; TNF concentration was assessed by ELISA. Cytokine levels are expressed as means of triplicate repeats ± SEM. Results shown combined data from 3 to 6 separate donors. Statistical analysis: student's t-test, ***p

    Techniques Used: Immunoprecipitation, Western Blot, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Transfection, Incubation, SDS Page, Concentration Assay

    37) Product Images from "Overexpression of TREM2 enhances glioma cell proliferation and invasion: a therapeutic target in human glioma"

    Article Title: Overexpression of TREM2 enhances glioma cell proliferation and invasion: a therapeutic target in human glioma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6221

    Mechanisms of TREM2 exert their functions in glioma cells Enrichment plots of gene expression signatures for KEGG apoptosis A. , Cromer invasion B. and KEGG chemokine pathways C. according to TREM2 expression levels. D. , E. , F. Protein levels of apoptosis-related factors (cleaved caspase 3 and Bad), anti-apoptosis (Bcl2), invasion (MMP2 and MMP9) and chemokine pathway related factors (CXCL10 and CXCR3) in glioma and normal tissues, and glioma cell lines (U87 and U373 cells) were detected by western blot. GAPDH was also detected as the control of sample loading. Representative western blots (left panel) and quantitative results were shown (right panel). Data were based on at least three independent experiments, and shown as the mean ± SD (* P
    Figure Legend Snippet: Mechanisms of TREM2 exert their functions in glioma cells Enrichment plots of gene expression signatures for KEGG apoptosis A. , Cromer invasion B. and KEGG chemokine pathways C. according to TREM2 expression levels. D. , E. , F. Protein levels of apoptosis-related factors (cleaved caspase 3 and Bad), anti-apoptosis (Bcl2), invasion (MMP2 and MMP9) and chemokine pathway related factors (CXCL10 and CXCR3) in glioma and normal tissues, and glioma cell lines (U87 and U373 cells) were detected by western blot. GAPDH was also detected as the control of sample loading. Representative western blots (left panel) and quantitative results were shown (right panel). Data were based on at least three independent experiments, and shown as the mean ± SD (* P

    Techniques Used: Expressing, Western Blot

    38) Product Images from "Proteomic Analysis of Apis cerana and Apis mellifera Larvae Fed with Heterospecific Royal Jelly and by CSBV Challenge"

    Article Title: Proteomic Analysis of Apis cerana and Apis mellifera Larvae Fed with Heterospecific Royal Jelly and by CSBV Challenge

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0102663

    Western blot Analysis of differentially expressed proteins. Six proteins were selected. Proteins were separated on a 12% SDS-PAGE gel and immunoblotted with anti-GAPDH, anti-PKG2, anti-Tpi, anti-FAH, anti-SOD, and anti-FBA. β-actin was used as an internal control. (A) The Western blot images of GAPDH, PKG2, TPI, FAH, SOD, FBA and Actin. The protein spots from Figure 2 . (B) Quantification of target protein expression.The relative fold change of GAPDH, PKG2, Tpi, FAH, SOD, FBA (normalized by Actin) was visulized by using Quantity One Software. The expression of GAPDH, SOD, FBA and Tpi were up-regulatedd. On the other hand, FAH and PGK were down-regulated. Error bar is standard deviation.
    Figure Legend Snippet: Western blot Analysis of differentially expressed proteins. Six proteins were selected. Proteins were separated on a 12% SDS-PAGE gel and immunoblotted with anti-GAPDH, anti-PKG2, anti-Tpi, anti-FAH, anti-SOD, and anti-FBA. β-actin was used as an internal control. (A) The Western blot images of GAPDH, PKG2, TPI, FAH, SOD, FBA and Actin. The protein spots from Figure 2 . (B) Quantification of target protein expression.The relative fold change of GAPDH, PKG2, Tpi, FAH, SOD, FBA (normalized by Actin) was visulized by using Quantity One Software. The expression of GAPDH, SOD, FBA and Tpi were up-regulatedd. On the other hand, FAH and PGK were down-regulated. Error bar is standard deviation.

    Techniques Used: Western Blot, SDS Page, Expressing, Software, Standard Deviation

    39) Product Images from "rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration"

    Article Title: rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-8-44

    Soluble and insoluble α-synuclein levels increase over time upon delivery of rAAV2/7-α-synuclein WT or A53T. (A) Western blot and (B) quantitative analysis (n = 3) of soluble α-synuclein in the cytoplasmic (TBS soluble) fraction of SN mouse lysates injected with rAAV2/7-eGFP (4 weeks time point) or rAAV2/7-α-synuclein WT or A53T (5 days, 2 weeks and 4 weeks time points). (C) Immunoblotting and (D) quantification (n = 3) of phosphorylated and detergent-insoluble α-synuclein in the Urea soluble fraction of injected mouse nigral homogenates. For detection, immunoblots for α-synuclein were performed using a panel of different antibodies against: mouse and human α-synuclein (Syn1), specific human α-synuclein (15G7) and P-S129 α-synuclein. GAPDH and β-actin serve as internal loading control in the TBS and Urea fractions, respectively.
    Figure Legend Snippet: Soluble and insoluble α-synuclein levels increase over time upon delivery of rAAV2/7-α-synuclein WT or A53T. (A) Western blot and (B) quantitative analysis (n = 3) of soluble α-synuclein in the cytoplasmic (TBS soluble) fraction of SN mouse lysates injected with rAAV2/7-eGFP (4 weeks time point) or rAAV2/7-α-synuclein WT or A53T (5 days, 2 weeks and 4 weeks time points). (C) Immunoblotting and (D) quantification (n = 3) of phosphorylated and detergent-insoluble α-synuclein in the Urea soluble fraction of injected mouse nigral homogenates. For detection, immunoblots for α-synuclein were performed using a panel of different antibodies against: mouse and human α-synuclein (Syn1), specific human α-synuclein (15G7) and P-S129 α-synuclein. GAPDH and β-actin serve as internal loading control in the TBS and Urea fractions, respectively.

    Techniques Used: Western Blot, Injection

    40) Product Images from "Multiple mRNA Decapping Enzymes in Mammalian Cells"

    Article Title: Multiple mRNA Decapping Enzymes in Mammalian Cells

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2010.10.010

    Nudt16 mRNA and Protein are Expressed in All Mouse Tissues Tested Nudt16 mRNA ( A ) and protein ( B ) levels within the indicated organ extracts were detected by Northern blot (20μg) and Western blot (100μg) analysis respectively. Lanes containing 50μg extract from human erythroleukemia K562 cells and murine erythroleukemia MEL cells are denoted. GAPDH was used as loading control. (C) The Nudt16 protein levels in 60μg extract from WT or Dcp2 β/β MEF cells were tested by Western Blot and Tubulin was used as a loading control.
    Figure Legend Snippet: Nudt16 mRNA and Protein are Expressed in All Mouse Tissues Tested Nudt16 mRNA ( A ) and protein ( B ) levels within the indicated organ extracts were detected by Northern blot (20μg) and Western blot (100μg) analysis respectively. Lanes containing 50μg extract from human erythroleukemia K562 cells and murine erythroleukemia MEL cells are denoted. GAPDH was used as loading control. (C) The Nudt16 protein levels in 60μg extract from WT or Dcp2 β/β MEF cells were tested by Western Blot and Tubulin was used as a loading control.

    Techniques Used: Northern Blot, Western Blot

    Nudt16 is a Cytoplasm mRNA Decapping Enzyme ( A ) Decapping activity of the recombinant His tagged human Dcp2, Nudt16 and mouse Nudt1, 4, 5, 7 and Nudt16L1 proteins are shown. Decapping products resolve on PEI-TLC developed in 0.45 M (NH 4 ) 2 SO 4 . ( B ) 293T cells cytoplasm and nuclear proteins were fractionated, extract from equal cell numbers were resolved on SDS PAGE and the distribution of Nudt16 protein was tested by Western Blot analysis. hnRNP C1/C2 and GAPDH were used as nuclear and cytoplasmic markers respectively. ( C ) FLAG-hNudt16 (Top) or hNudt16-FLAG (Bottom) was overexpressed in U2OS cells. The epitope tagged protein was localized by indirect immunofluorescence with an anti-FLAG antibody by confocal microscopy. The presented images are representative of > 95% of the transfected cells. The Differential Interference Contrast (DIC) images of the same cells are shown as indicated.
    Figure Legend Snippet: Nudt16 is a Cytoplasm mRNA Decapping Enzyme ( A ) Decapping activity of the recombinant His tagged human Dcp2, Nudt16 and mouse Nudt1, 4, 5, 7 and Nudt16L1 proteins are shown. Decapping products resolve on PEI-TLC developed in 0.45 M (NH 4 ) 2 SO 4 . ( B ) 293T cells cytoplasm and nuclear proteins were fractionated, extract from equal cell numbers were resolved on SDS PAGE and the distribution of Nudt16 protein was tested by Western Blot analysis. hnRNP C1/C2 and GAPDH were used as nuclear and cytoplasmic markers respectively. ( C ) FLAG-hNudt16 (Top) or hNudt16-FLAG (Bottom) was overexpressed in U2OS cells. The epitope tagged protein was localized by indirect immunofluorescence with an anti-FLAG antibody by confocal microscopy. The presented images are representative of > 95% of the transfected cells. The Differential Interference Contrast (DIC) images of the same cells are shown as indicated.

    Techniques Used: Activity Assay, Recombinant, Thin Layer Chromatography, SDS Page, Western Blot, Immunofluorescence, Confocal Microscopy, Transfection

    Dcp2 Protein is Differentially Expressed in Mammalian Organs ( A ) Dcp2 protein is not detectable in a subset of organ extracts. Protein extract (100 μg) from the indicated organs were resolved by SDS-polyacrylamide gel electrophoresis and Dcp2 protein was detected by Western blot analysis using GAPDH as a loading control. Fifty micrograms K562 extract was used as a positive control and migration of each protein is denoted on the right. ( B ) Extract from the indicated human organs were resolved and analyzed as in A except eIF4E was used as a loading control. The brain extract is from brain cortex. Fifty micrograms HeLa S15 extract and 293T cell total extract were used as positive controls. ( C ) The presence of Dcp2 was detected as in A from the indicated mouse extracts derived from embryonic day 14.5 (E14.5), embryonic day 16.5 (E16.5), postnatal day 0 (P0) and 8 weeks old adults (Adult).
    Figure Legend Snippet: Dcp2 Protein is Differentially Expressed in Mammalian Organs ( A ) Dcp2 protein is not detectable in a subset of organ extracts. Protein extract (100 μg) from the indicated organs were resolved by SDS-polyacrylamide gel electrophoresis and Dcp2 protein was detected by Western blot analysis using GAPDH as a loading control. Fifty micrograms K562 extract was used as a positive control and migration of each protein is denoted on the right. ( B ) Extract from the indicated human organs were resolved and analyzed as in A except eIF4E was used as a loading control. The brain extract is from brain cortex. Fifty micrograms HeLa S15 extract and 293T cell total extract were used as positive controls. ( C ) The presence of Dcp2 was detected as in A from the indicated mouse extracts derived from embryonic day 14.5 (E14.5), embryonic day 16.5 (E16.5), postnatal day 0 (P0) and 8 weeks old adults (Adult).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, Positive Control, Migration, Derivative Assay

    Related Articles

    CtB Assay:

    Article Title: GM1 ganglioside-independent intoxication by Cholera toxin
    Article Snippet: .. Blots were incubated in mild stripping buffer (200 mM glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) at 37°C for 45 min and incubated with anti-histone H3 antibody (Abcam, Cat. No. ab1791, 1:2000 dilution, for AAL blot), anti-α-tubulin antibody (Sigma-Aldrich, Cat. No. T6199, 1:10000 dilution, for PNA and CTB blot), or anti-GAPDH antibody (Abcam, Cat. No. ab8245, 1:10000 dilution, for streptavidin-POD blot) at room temperature for 1 h and washed with TBST. .. The blots were then incubated with HRP conjugated goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, Cat. No. 65–6120, 1:5000 dilution) or HRP conjugated goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific, Cat. No. 62–6520, 1:5000 dilution) at room temperature for 1 h. All blots were developed with SuperSignal West Pico Chemiluminescence Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat. No. 34095) with a ChemiDoc MP Imaging system.

    Blocking Assay:

    Article Title: circSMAD2 inhibits the epithelial–mesenchymal transition by targeting miR-629 in hepatocellular carcinoma
    Article Snippet: .. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies: anti-SMAD2 (Abcam, Cambridge, MA, USA), anti-GAPDH (Abcam), anti-E-cadherin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Cell Signaling Technology), anti-Snail (Cell Signaling Technology), and anti-Vimentin (Cell Signaling Technology). .. Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies.

    Article Title: The combination of NVP-BEZ235 and rapamycin regulates nasopharyngeal carcinoma cell viability and apoptosis via the PI3K/AKT/mTOR pathway
    Article Snippet: .. Following blocking with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature for 1 h, the membranes were probed with the following mouse monoclonal antibodies: Anti-PI3K (ab86714; 1:1,000), anti-AKT (ab175354; 1:1,000), anti-phosphorylated (p)-AKT (ab105731; 1:500), anti-mTOR (ab87540; 1:1,000), anti-GAPDH (AB8245; 1:1,000) and rabbit monoclonal anti-p-mTOR (ab109268; 1:1,000; all Abcam, Cambridge, MA, USA) at 4°C overnight. .. The membranes were incubated with the rabbit anti-mouse IgG (ab6728; 1:10,000) or goat anti-rabbit IgG (ab205718; 1:10,000) horseradish peroxidase-labeled secondary antibodies (both Abcam) at room temperature for 1 h. Following washing with TBST, the blot was examined with electrochemiluminescence solution (Pierce; Thermo Fisher Scientific, Inc.).

    Concentration Assay:

    Article Title: Inhibition of Ep3 attenuates migration and promotes apoptosis of non-small cell lung cancer cells via suppression of TGF-β/Smad signaling
    Article Snippet: .. Membranes were blocked in 5% bovine serum albumin (Sigma Aldrich; Merck KGaA) at room temperature for 1 h, and subsequently immunoblotted with the following primary antibodies according to the recommended dilution concentration: Anti-Ep3 (1:500; cat. no. P8372; Sigma-Aldrich; Merck KGaA), anti-caspase-3 (1:1,000; cat. no. 9662), anti-B cell lymphoma (Bcl)-2 (1:1,000; cat. no. 2872), anti-Bcl-associated × protein (Bax; 1:1,000; cat. no. 2772; all Cell Signaling Technology, Inc., Danvers, MA, USA), anti-MMP-9 (1:500; cat. no. ab58803; Abcam, Cambridge, UK), anti-VEGF (1:1,000; cat. no. V6627; Sigma-Aldrich; Meck KGaA), anti-TGF-β1 (1:1,000; cat. no. SAB4502954; Sigma-Aldrich; Merck KGaA), anti-Smad-2 (1:1,000; cat. no. ab63576), anti-Smad-3 (1:2,000; cat. no. ab40854), anti-phosphorylated (p)-Smad2 (1:800; cat. no. ab53100), anti-p-Smad3 (1:2,000; cat. no. ab52903) and anti-GAPDH (1:2,000; cat. no. ab8245; all Abcam) at 4°C overnight. .. Then, membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies: Anti-rabbit IgG (1:10,000; cat. no. 7074), anti-mouse IgG (1:10,000; cat. no. 7076; both Cell Signaling Technology, Inc.) and donkey anti-goat IgG (1:10,000; cat. no. ab6885; Abcam) at room temperature for 1 h. GAPDH was used as the loading control.

    Incubation:

    Article Title: GM1 ganglioside-independent intoxication by Cholera toxin
    Article Snippet: .. Blots were incubated in mild stripping buffer (200 mM glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) at 37°C for 45 min and incubated with anti-histone H3 antibody (Abcam, Cat. No. ab1791, 1:2000 dilution, for AAL blot), anti-α-tubulin antibody (Sigma-Aldrich, Cat. No. T6199, 1:10000 dilution, for PNA and CTB blot), or anti-GAPDH antibody (Abcam, Cat. No. ab8245, 1:10000 dilution, for streptavidin-POD blot) at room temperature for 1 h and washed with TBST. .. The blots were then incubated with HRP conjugated goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, Cat. No. 65–6120, 1:5000 dilution) or HRP conjugated goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific, Cat. No. 62–6520, 1:5000 dilution) at room temperature for 1 h. All blots were developed with SuperSignal West Pico Chemiluminescence Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat. No. 34095) with a ChemiDoc MP Imaging system.

    Article Title: circSMAD2 inhibits the epithelial–mesenchymal transition by targeting miR-629 in hepatocellular carcinoma
    Article Snippet: .. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies: anti-SMAD2 (Abcam, Cambridge, MA, USA), anti-GAPDH (Abcam), anti-E-cadherin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Cell Signaling Technology), anti-Snail (Cell Signaling Technology), and anti-Vimentin (Cell Signaling Technology). .. Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies.

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling
    Article Snippet: .. Blots were probed with specific primary antibodies against LIF (Abcam, ab135629, 1:500), LIFR (Santa Cruz Biotechnology, sc-515337, 1:500), phospho-YAP1 (Cell Signaling, 13008, 1:2000), YAP1 (Cell Signaling, 14074, 1:2000), phospho-FAK (Invitrogen, 700255, 1:5000), FAK (Santa Cruz Biotechnology, sc-558, 1:500), PECAM-1 (CD31) (Santa Cruz Biotechnology, sc-376764, 1:500), VE-cad (Santa Cruz Biotechnology, sc-9989, 1:1000), E-cad (BD Transduction Laboratories, 610181, 1:5000), phospho-PXN (Y118) (Abcam, ab109547, 1:4000), PXN (BD Transduction Laboratories, 610052, 1:10,000), phospho-p70S6K1 (T389) (Cell Signaling, 9234, 1:2000), p70S6K1 (Abcam, ab32529, 1:10,000), N-cad (Abcam, ab76011, 1:10,000), VIM (Sigma-Aldrich, V5255, 1:500), IQGAP1 (Santa Cruz Biotechnology, sc-81906, 1:1000), phospho-SRC (Cell Signaling, 6942, 1:2000), SRC (Cell Signaling, 2109, 1:2000), TKS5 (Proteintech, 18976-1-AP, 1:1500), CTTN (Abcam, ab81208, 1:10,000), MMP2 (Proteintech, 10373-2-AP, 1:800), and GAPDH (Abcam, ab8245, 1:5000) by incubation with horseradish peroxidase-conjugated secondary antibody and developed with enhanced chemiluminescence detection reagent (GE Healthcare). ..

    Stripping Membranes:

    Article Title: GM1 ganglioside-independent intoxication by Cholera toxin
    Article Snippet: .. Blots were incubated in mild stripping buffer (200 mM glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) at 37°C for 45 min and incubated with anti-histone H3 antibody (Abcam, Cat. No. ab1791, 1:2000 dilution, for AAL blot), anti-α-tubulin antibody (Sigma-Aldrich, Cat. No. T6199, 1:10000 dilution, for PNA and CTB blot), or anti-GAPDH antibody (Abcam, Cat. No. ab8245, 1:10000 dilution, for streptavidin-POD blot) at room temperature for 1 h and washed with TBST. .. The blots were then incubated with HRP conjugated goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, Cat. No. 65–6120, 1:5000 dilution) or HRP conjugated goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific, Cat. No. 62–6520, 1:5000 dilution) at room temperature for 1 h. All blots were developed with SuperSignal West Pico Chemiluminescence Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat. No. 34095) with a ChemiDoc MP Imaging system.

    Western Blot:

    Article Title: Signal transducer and activator of transcription 3 signaling upregulates fascin via nuclear factor-?B in gastric cancer: Implications in cell invasion and migration
    Article Snippet: .. Antibodies used for western blotting and chromatin immunoprecipitation (ChIP) assays were anti-phosphotyrosine (p)STAT3 (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-STAT3 (Cell Signaling Technology, Inc.), anti-nuclear factor (NF)-κB p50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-fascin (Abcam, Cambridge, UK), anti-hairy and enhancer of split-1 (Hes-1; Abcam), anti-activated-Notch1 (Abcam), anti-activated-Notch2 (Abcam), anti-matrix metalloproteinase (MMP)-2 and anti-MMP-9 (Cell Signaling Technology, Inc.), anti-GAPDH (Abcam), anti-rabbit immunoglobulin (Ig)G and horseradish peroxidase-linked antibody (Cell Signaling Technology, Inc.). .. ChIP assays ChIP assays were performed using a ChIP assay kit, according to the manufacturer’s instructions (Upstate Biotechnology, Lake Placid, NY, USA) as described previously ( , ).

    Chromatin Immunoprecipitation:

    Article Title: Signal transducer and activator of transcription 3 signaling upregulates fascin via nuclear factor-?B in gastric cancer: Implications in cell invasion and migration
    Article Snippet: .. Antibodies used for western blotting and chromatin immunoprecipitation (ChIP) assays were anti-phosphotyrosine (p)STAT3 (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-STAT3 (Cell Signaling Technology, Inc.), anti-nuclear factor (NF)-κB p50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-fascin (Abcam, Cambridge, UK), anti-hairy and enhancer of split-1 (Hes-1; Abcam), anti-activated-Notch1 (Abcam), anti-activated-Notch2 (Abcam), anti-matrix metalloproteinase (MMP)-2 and anti-MMP-9 (Cell Signaling Technology, Inc.), anti-GAPDH (Abcam), anti-rabbit immunoglobulin (Ig)G and horseradish peroxidase-linked antibody (Cell Signaling Technology, Inc.). .. ChIP assays ChIP assays were performed using a ChIP assay kit, according to the manufacturer’s instructions (Upstate Biotechnology, Lake Placid, NY, USA) as described previously ( , ).

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    Abcam gapdh
    Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. <t>GAPDH</t> was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). <t>CTTN</t> ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm
    Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh
    SENP2 suppresses the Notch and NF-κB signaling pathways. (A) SENP2-overexpressingMEC2 cells and the control cells were cultured in 6 cm dishes and they were harvested after reaching 80-90% confluence, and these cells were then examind by western blot analysis to detect the expression levels of IKKβ, <t>IKKα,</t> IκBα, Cyld, C-myc, β-catenin, Notch1, p50, p65 and SENP2. (B) MEC2 cells in which SENP2 was silenced and the control cells were treated as described in (A). <t>GAPDH</t> was used to confirm equal amount of proteins loaded in each lane. NC-OE-SENP2, MEC2 cells transfected with null (control) overexpression vector; OE-SENP2, MEC2 cells transfected with SENP2 overexpression vector; NC-shRNA-SENP2, MEC2 cells transfected with null control shRNA; shRNA-SENP2, MEC2 cells transfected with shRNA against SENP2.
    Anti Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. GAPDH was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). CTTN ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. GAPDH was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). CTTN ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm

    Article Snippet: Blots were probed with specific primary antibodies against LIF (Abcam, ab135629, 1:500), LIFR (Santa Cruz Biotechnology, sc-515337, 1:500), phospho-YAP1 (Cell Signaling, 13008, 1:2000), YAP1 (Cell Signaling, 14074, 1:2000), phospho-FAK (Invitrogen, 700255, 1:5000), FAK (Santa Cruz Biotechnology, sc-558, 1:500), PECAM-1 (CD31) (Santa Cruz Biotechnology, sc-376764, 1:500), VE-cad (Santa Cruz Biotechnology, sc-9989, 1:1000), E-cad (BD Transduction Laboratories, 610181, 1:5000), phospho-PXN (Y118) (Abcam, ab109547, 1:4000), PXN (BD Transduction Laboratories, 610052, 1:10,000), phospho-p70S6K1 (T389) (Cell Signaling, 9234, 1:2000), p70S6K1 (Abcam, ab32529, 1:10,000), N-cad (Abcam, ab76011, 1:10,000), VIM (Sigma-Aldrich, V5255, 1:500), IQGAP1 (Santa Cruz Biotechnology, sc-81906, 1:1000), phospho-SRC (Cell Signaling, 6942, 1:2000), SRC (Cell Signaling, 2109, 1:2000), TKS5 (Proteintech, 18976-1-AP, 1:1500), CTTN (Abcam, ab81208, 1:10,000), MMP2 (Proteintech, 10373-2-AP, 1:800), and GAPDH (Abcam, ab8245, 1:5000) by incubation with horseradish peroxidase-conjugated secondary antibody and developed with enhanced chemiluminescence detection reagent (GE Healthcare).

    Techniques: Migration, Imaging, Immunohistochemistry, Expressing, Staining, Western Blot, Immunofluorescence

    Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: Gastrodin protects MC3T3-E1 osteoblasts from dexamethasone-induced cellular dysfunction and promotes bone formation via induction of the NRF2 signaling pathway

    doi: 10.3892/ijmm.2018.3414

    Figure Lengend Snippet: Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Article Snippet: Rabbit anti-HO-1 (cat. no. ab68477; 1:1,000), rabbit anti-NQO-1 (cat. no. ab76956; 1:1,000), and mouse anti-β-actin (cat. no. ab8245; 1:1,000), Anti-OCN (cat. no. ab93876; 1:1,000) monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Standard Deviation

    QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Journal: OncoTargets and therapy

    Article Title: circSMAD2 inhibits the epithelial–mesenchymal transition by targeting miR-629 in hepatocellular carcinoma

    doi: 10.2147/OTT.S158008

    Figure Lengend Snippet: QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies: anti-SMAD2 (Abcam, Cambridge, MA, USA), anti-GAPDH (Abcam), anti-E-cadherin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Cell Signaling Technology), anti-Snail (Cell Signaling Technology), and anti-Vimentin (Cell Signaling Technology).

    Techniques: Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Negative Control

    SENP2 suppresses the Notch and NF-κB signaling pathways. (A) SENP2-overexpressingMEC2 cells and the control cells were cultured in 6 cm dishes and they were harvested after reaching 80-90% confluence, and these cells were then examind by western blot analysis to detect the expression levels of IKKβ, IKKα, IκBα, Cyld, C-myc, β-catenin, Notch1, p50, p65 and SENP2. (B) MEC2 cells in which SENP2 was silenced and the control cells were treated as described in (A). GAPDH was used to confirm equal amount of proteins loaded in each lane. NC-OE-SENP2, MEC2 cells transfected with null (control) overexpression vector; OE-SENP2, MEC2 cells transfected with SENP2 overexpression vector; NC-shRNA-SENP2, MEC2 cells transfected with null control shRNA; shRNA-SENP2, MEC2 cells transfected with shRNA against SENP2.

    Journal: International Journal of Oncology

    Article Title: SENP2 exerts an anti-tumor effect on chronic lymphocytic leukemia cells through the inhibition of the Notch and NF-κB signaling pathways

    doi: 10.3892/ijo.2018.4635

    Figure Lengend Snippet: SENP2 suppresses the Notch and NF-κB signaling pathways. (A) SENP2-overexpressingMEC2 cells and the control cells were cultured in 6 cm dishes and they were harvested after reaching 80-90% confluence, and these cells were then examind by western blot analysis to detect the expression levels of IKKβ, IKKα, IκBα, Cyld, C-myc, β-catenin, Notch1, p50, p65 and SENP2. (B) MEC2 cells in which SENP2 was silenced and the control cells were treated as described in (A). GAPDH was used to confirm equal amount of proteins loaded in each lane. NC-OE-SENP2, MEC2 cells transfected with null (control) overexpression vector; OE-SENP2, MEC2 cells transfected with SENP2 overexpression vector; NC-shRNA-SENP2, MEC2 cells transfected with null control shRNA; shRNA-SENP2, MEC2 cells transfected with shRNA against SENP2.

    Article Snippet: The anti-IKKα (1:1,000; cat. no. ab32041) and anti-GAPDH (1:1,000; cat. no. ab37168) antibodies were from Abcam (Cambridge, UK).

    Techniques: Cell Culture, Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, shRNA