anti gapdh  (Millipore)


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    Anti GAPDH
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    Millipore anti gapdh
    Anti GAPDH

    https://www.bioz.com/result/anti gapdh/product/Millipore
    Average 99 stars, based on 1300 article reviews
    Price from $9.99 to $1999.99
    anti gapdh - by Bioz Stars, 2020-09
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    1) Product Images from "Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP, et al. Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP"

    Article Title: Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP, et al. Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP

    Journal: Cell Proliferation

    doi: 10.1111/cpr.12833

    Both TCF21 and HHIP were target genes of miR‐25‐3p. A, The mRNA expression of TCF21 and HHIP in CHB patients with PNALT (≥A2) was detected by RT‐PCR assay. B, Co‐immunoprecipitation experiments indicated that TCF21 directly interact with HHIP in HepG2.2.15 cells. C, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in TCF21 inhibition or overexpression transfected HepG2.2.15 cells. D, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in HHIP inhibition or overexpression transfected HepG2.2.15 cells. E, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 inhibition or HHIP inhibition transfected HepG2.2.15 cells. F, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 overexpression or HHIP overexpression transfected HepG2.2.15 cells. G, TargetScan database showed that binding site of TCF21 or HHIP and miR‐340‐5p. H, Luciferase reporter assays were used to prove that miR‐340‐5p can target TCF21 or HHIP. I, J, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in miR‐25‐3p mimics or miR‐25‐3p inhibitor‐transfected HepG2.2.15 cells. K, Correlation analysis of TCF21/HHIP and miR‐25‐3p in CHB patients with PNALT (≥A2), correlation analysis of TCF21 and HHIP/miR‐25‐3p in HBV‐positive patients with liver cancer and correlation analysis of TCF21 and HHIP/miR‐25‐3p in CHB patients with PNALT (≥A2). GAPDH or U6 was used as a load control. Data are presented as the mean ± standard deviation. ** P
    Figure Legend Snippet: Both TCF21 and HHIP were target genes of miR‐25‐3p. A, The mRNA expression of TCF21 and HHIP in CHB patients with PNALT (≥A2) was detected by RT‐PCR assay. B, Co‐immunoprecipitation experiments indicated that TCF21 directly interact with HHIP in HepG2.2.15 cells. C, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in TCF21 inhibition or overexpression transfected HepG2.2.15 cells. D, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in HHIP inhibition or overexpression transfected HepG2.2.15 cells. E, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 inhibition or HHIP inhibition transfected HepG2.2.15 cells. F, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 overexpression or HHIP overexpression transfected HepG2.2.15 cells. G, TargetScan database showed that binding site of TCF21 or HHIP and miR‐340‐5p. H, Luciferase reporter assays were used to prove that miR‐340‐5p can target TCF21 or HHIP. I, J, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in miR‐25‐3p mimics or miR‐25‐3p inhibitor‐transfected HepG2.2.15 cells. K, Correlation analysis of TCF21/HHIP and miR‐25‐3p in CHB patients with PNALT (≥A2), correlation analysis of TCF21 and HHIP/miR‐25‐3p in HBV‐positive patients with liver cancer and correlation analysis of TCF21 and HHIP/miR‐25‐3p in CHB patients with PNALT (≥A2). GAPDH or U6 was used as a load control. Data are presented as the mean ± standard deviation. ** P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Inhibition, Over Expression, Transfection, Western Blot, Binding Assay, Luciferase, Standard Deviation

    The functions of miR‐25‐3p inhibitors abolishing the effects of CHB‐PNALT‐Exo (≥A2) on the proliferation and metastasis were reversed by knockdown of TCF21 and HHIP in HepG2.2.15 cells. After siRNA‐TCF21 and siRNA‐HHIP were transfected into CHB‐PNALT‐Exo (≥A2) containing miR‐25‐3p inhibitor‐treated HepG2.2.15 cells, cell viability, apoptosis, invasion, migration and the expression of cleaved caspase‐3/‐9, Ki67 and E‐cadherin were detected by CCK‐8 assay (A), flow cytometry analysis (B), clone formation assay (C), transwell assay (D), scratch‐wound assay (E) and Western blot assay (F). GAPDH was used as a load control. Data are presented as the mean ± standard deviation. ** P
    Figure Legend Snippet: The functions of miR‐25‐3p inhibitors abolishing the effects of CHB‐PNALT‐Exo (≥A2) on the proliferation and metastasis were reversed by knockdown of TCF21 and HHIP in HepG2.2.15 cells. After siRNA‐TCF21 and siRNA‐HHIP were transfected into CHB‐PNALT‐Exo (≥A2) containing miR‐25‐3p inhibitor‐treated HepG2.2.15 cells, cell viability, apoptosis, invasion, migration and the expression of cleaved caspase‐3/‐9, Ki67 and E‐cadherin were detected by CCK‐8 assay (A), flow cytometry analysis (B), clone formation assay (C), transwell assay (D), scratch‐wound assay (E) and Western blot assay (F). GAPDH was used as a load control. Data are presented as the mean ± standard deviation. ** P

    Techniques Used: Transfection, Migration, Expressing, CCK-8 Assay, Flow Cytometry, Tube Formation Assay, Transwell Assay, Scratch Wound Assay Assay, Western Blot, Standard Deviation

    2) Product Images from "Long QT syndrome caveolin‐3 mutations differentially modulate Kv4 and Cav1.2 channels to contribute to action potential prolongation"

    Article Title: Long QT syndrome caveolin‐3 mutations differentially modulate Kv4 and Cav1.2 channels to contribute to action potential prolongation

    Journal: The Journal of Physiology

    doi: 10.1113/JP276014

    Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.
    Figure Legend Snippet: Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.

    Techniques Used: Expressing, Transfection, Confocal Microscopy, Western Blot, Plasmid Preparation, Negative Control

    3) Product Images from "Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers"

    Article Title: Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers

    Journal: Autophagy

    doi: 10.4161/15548627.2014.981788

    For figure legend, see page 2133. Figure 5 ( See previous page ). The transcription co-activator YAP1 is a key player in the autophagy-dependent proliferation and invasion of TN BC cells. ( A ) Western blots showing phosphorylated-YAP1 (P-YAP1), YAP1, and AP2A1 proteins in stable cell lines following 3D culture. AP2A1 is used as internal control for protein loading. The bar graph (right panel) shows the corresponding quantification of P-YAP1/YAP1 protein level ratios. ( B ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3D culture. GAPDH is used as an internal control for total mRNA expression. ( C ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with 3-methyladenine (3-MA; 20 mM) for 3 h. ( D ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3-MA treatment. GAPDH is used as an internal control for total mRNA expression. ( E ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with rapamycin (2 μM) for 3 h. ( F ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following rapamycin treatment. GAPDH is used as an internal control for total mRNA expression. ( G ) Left, western blots showing YAP1 protein levels in MDA231 cells after transfection with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. AP2A1 is used as an internal control for protein loading. Middle, representative bright field images from Control- or YAP1-depleted cells cultured in 3D, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Right, the bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( H ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from MDA231 cells transiently transfected with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. for 72 h prior to the assay. Left histogram: Numbers of invading cells, which passed through a Transwell over 6 h of incubation. Right panel: Percentage of cells, relative to siCtrl (100%), which passed through a Transwell over 6 h of incubation. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( I ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from autophagy-deficient MDA231 cells (36 h of transfection with si ATG7 or si ATG5 , as indicated), transfected again (36 h) with an empty vector or a vector expressing YAP1-S127A, a nonphosphorylable mutant form of YAP1. Numbers of invading cells, which passed through a Transwell over 6 h of incubation, are shown. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( J ) Results are from autophagy-proficient (sh Ctrl ) or autophagy-deficient (sh ATG5 ) MDA231 cells, transfected with an empty vector or a vector expressing YAP1-S127A, after 3 d of 3D culture. Representative bright field images from ATG5-depleted cells expressing or not YAP1-S127A, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 5 independent experiments). P -values are based on the Student t test.
    Figure Legend Snippet: For figure legend, see page 2133. Figure 5 ( See previous page ). The transcription co-activator YAP1 is a key player in the autophagy-dependent proliferation and invasion of TN BC cells. ( A ) Western blots showing phosphorylated-YAP1 (P-YAP1), YAP1, and AP2A1 proteins in stable cell lines following 3D culture. AP2A1 is used as internal control for protein loading. The bar graph (right panel) shows the corresponding quantification of P-YAP1/YAP1 protein level ratios. ( B ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3D culture. GAPDH is used as an internal control for total mRNA expression. ( C ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with 3-methyladenine (3-MA; 20 mM) for 3 h. ( D ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3-MA treatment. GAPDH is used as an internal control for total mRNA expression. ( E ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with rapamycin (2 μM) for 3 h. ( F ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following rapamycin treatment. GAPDH is used as an internal control for total mRNA expression. ( G ) Left, western blots showing YAP1 protein levels in MDA231 cells after transfection with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. AP2A1 is used as an internal control for protein loading. Middle, representative bright field images from Control- or YAP1-depleted cells cultured in 3D, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Right, the bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( H ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from MDA231 cells transiently transfected with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. for 72 h prior to the assay. Left histogram: Numbers of invading cells, which passed through a Transwell over 6 h of incubation. Right panel: Percentage of cells, relative to siCtrl (100%), which passed through a Transwell over 6 h of incubation. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( I ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from autophagy-deficient MDA231 cells (36 h of transfection with si ATG7 or si ATG5 , as indicated), transfected again (36 h) with an empty vector or a vector expressing YAP1-S127A, a nonphosphorylable mutant form of YAP1. Numbers of invading cells, which passed through a Transwell over 6 h of incubation, are shown. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( J ) Results are from autophagy-proficient (sh Ctrl ) or autophagy-deficient (sh ATG5 ) MDA231 cells, transfected with an empty vector or a vector expressing YAP1-S127A, after 3 d of 3D culture. Representative bright field images from ATG5-depleted cells expressing or not YAP1-S127A, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 5 independent experiments). P -values are based on the Student t test.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, Stable Transfection, Quantitative RT-PCR, Expressing, Cell Culture, Transfection, Invasion Assay, Incubation, Plasmid Preparation, Mutagenesis

    4) Product Images from "hCAF1/CNOT7 regulates interferon signalling by targeting STAT1"

    Article Title: hCAF1/CNOT7 regulates interferon signalling by targeting STAT1

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2013.11

    Constitutive recruitment of STAT1 at a subset of STAT1-target promoters in hCAF1 knockdown cells. ChIP assays of untreated hCAF1 kd and control cells were performed using antibodies anti-STAT1 ( A ) and anti-acetyl H4 ( B ). Enriched DNA fragments were quantified by qPCR using specific primers for the indicated promoters with respect to the input DNA and normalized to a reference locus (3′ downstream region of the GAPDH gene). Rabbit IgGs were used as a negative control. ( C–E ) hCAF1 affects chromatin accessibility. ( C ) Schematic representation of GAS, PST1 site and primer positions on IFI27 promoter. ( D ) hCAF1 kd and control cells and ( E ) hCAF1 kd transfected with empty pCIflag (mock) or with pCIflag-mCAF1 (hCAF1 kd -mCAF1, rescued cells expressing mCAF1) were exposed or not to IFNγ for 6 h. Isolated nuclei were then treated with a limiting concentration of PST1 restriction enzyme, which cut GAS containing region in IFI27 promoter. DNA was then purified and the level of intact DNA was determined by qPCR using oligos flanking the GAS element or control region illustrated in ( C ). The experiments were performed in triplicate, expressed as mean values and are representative of at least three independent experiments. Standard deviations are shown.
    Figure Legend Snippet: Constitutive recruitment of STAT1 at a subset of STAT1-target promoters in hCAF1 knockdown cells. ChIP assays of untreated hCAF1 kd and control cells were performed using antibodies anti-STAT1 ( A ) and anti-acetyl H4 ( B ). Enriched DNA fragments were quantified by qPCR using specific primers for the indicated promoters with respect to the input DNA and normalized to a reference locus (3′ downstream region of the GAPDH gene). Rabbit IgGs were used as a negative control. ( C–E ) hCAF1 affects chromatin accessibility. ( C ) Schematic representation of GAS, PST1 site and primer positions on IFI27 promoter. ( D ) hCAF1 kd and control cells and ( E ) hCAF1 kd transfected with empty pCIflag (mock) or with pCIflag-mCAF1 (hCAF1 kd -mCAF1, rescued cells expressing mCAF1) were exposed or not to IFNγ for 6 h. Isolated nuclei were then treated with a limiting concentration of PST1 restriction enzyme, which cut GAS containing region in IFI27 promoter. DNA was then purified and the level of intact DNA was determined by qPCR using oligos flanking the GAS element or control region illustrated in ( C ). The experiments were performed in triplicate, expressed as mean values and are representative of at least three independent experiments. Standard deviations are shown.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Transfection, Expressing, Isolation, Concentration Assay, Purification

    5) Product Images from "Systemic activation of NLRP3 inflammasome and plasma α-synuclein levels are correlated with motor severity and progression in Parkinson’s disease"

    Article Title: Systemic activation of NLRP3 inflammasome and plasma α-synuclein levels are correlated with motor severity and progression in Parkinson’s disease

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-019-1670-6

    Expression of inflammasome-related genes in the PBMCs of PD patients and healthy controls. a – c NLRP3, NLRP1, and NLRC4 mRNA expression levels relative to GAPDH were determined by RT-qPCR analysis as described in the “ Methods ” section. d – f ASC, Caspase-1, and IL-1β mRNA expression relative to GAPDH were determined by RT-qPCR analysis. n = 24 for control, n = 43 for PD groups respectively. Data are expressed as mean ± SEM, one-way ANOVA
    Figure Legend Snippet: Expression of inflammasome-related genes in the PBMCs of PD patients and healthy controls. a – c NLRP3, NLRP1, and NLRC4 mRNA expression levels relative to GAPDH were determined by RT-qPCR analysis as described in the “ Methods ” section. d – f ASC, Caspase-1, and IL-1β mRNA expression relative to GAPDH were determined by RT-qPCR analysis. n = 24 for control, n = 43 for PD groups respectively. Data are expressed as mean ± SEM, one-way ANOVA

    Techniques Used: Expressing, Quantitative RT-PCR

    6) Product Images from "Cyclooxygenase-2 mediated synergistic effect of ursolic acid in combination with paclitaxel against human gastric carcinoma"

    Article Title: Cyclooxygenase-2 mediated synergistic effect of ursolic acid in combination with paclitaxel against human gastric carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21576

    Effects of UA and PTX on the COX-2, PCNA, Bcl-2, and Bax expression in BGC-823 and SGC-7901 cells Cells were treated with 12 μM UA, 2.5 nM PTX, or both for 48 hours. (A and C) COX-2, PCNA, Bcl-2, and Bax expression was detected by Western blot analysis. (B and D) The histogram represents the relative expressions of COX-2, PCNA, Bcl-2, and Bax compared with GAPDH. Each data point represents the mean ± SD from three independent experiments. * P
    Figure Legend Snippet: Effects of UA and PTX on the COX-2, PCNA, Bcl-2, and Bax expression in BGC-823 and SGC-7901 cells Cells were treated with 12 μM UA, 2.5 nM PTX, or both for 48 hours. (A and C) COX-2, PCNA, Bcl-2, and Bax expression was detected by Western blot analysis. (B and D) The histogram represents the relative expressions of COX-2, PCNA, Bcl-2, and Bax compared with GAPDH. Each data point represents the mean ± SD from three independent experiments. * P

    Techniques Used: Expressing, Western Blot

    7) Product Images from "Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells *"

    Article Title: Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.730226

    shRNA-mediated knockdown of caveolin-1 expression causes redistribution of the P2Y 2 R from membrane rafts. A, immunoblot analysis of hHAP2Y 2 R, Cav-1, and GAPDH (control) expression in serum-starved WT 1321N1 cells ( lane 1 ) or hHAP2Y 2 R 1321N1 cells ( lanes
    Figure Legend Snippet: shRNA-mediated knockdown of caveolin-1 expression causes redistribution of the P2Y 2 R from membrane rafts. A, immunoblot analysis of hHAP2Y 2 R, Cav-1, and GAPDH (control) expression in serum-starved WT 1321N1 cells ( lane 1 ) or hHAP2Y 2 R 1321N1 cells ( lanes

    Techniques Used: shRNA, Expressing

    Caveolin-1 knockdown inhibits P2Y 2 R-mediated increases in [Ca 2+ ] i in C6 glioma cells. A, representative immunoblot of P2Y 2 R, Cav-1, and GAPDH (control) expression in WT C6 cells ( 1st lane ), C6 cells after infection with scrambled (SCRAM) shRNA ( 2nd lane
    Figure Legend Snippet: Caveolin-1 knockdown inhibits P2Y 2 R-mediated increases in [Ca 2+ ] i in C6 glioma cells. A, representative immunoblot of P2Y 2 R, Cav-1, and GAPDH (control) expression in WT C6 cells ( 1st lane ), C6 cells after infection with scrambled (SCRAM) shRNA ( 2nd lane

    Techniques Used: Expressing, Infection, shRNA

    8) Product Images from "Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells"

    Article Title: Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113262

    TSA induces early differentiation of ESCs and promotes mesodermal lineage differentiation. ( A ) Bright-field images, alkaline phosphatase staining of ESCs and representative immunofluorescence images of Oct4 staining in control or TSA-treated ESCs (10 and 20 ng/ml) in the presence of LIF. ( B ) Western blotting verification of H3, acetyl-H3, H4, and acetyl-H4 in control or TSA-treated ESCs (10 and 20 ng/ml). GAPDH was used as a loading control. ( C ) The relative expression levels of Oct4, Nanog, and Rex1 mRNA in control or TSA-treated ESCs (10 and 20 ng/ml). ( D, E ) QRT-PCR analysis for marker genes of three germ layers (endoderm, mesoderm and ectoderm) in control or TSA-treated ESCs (10 and 20 ng/ml), under the monolayer differentiation condition without LIF. The cells were treated by TSA after removing LIF for 24h and collected mRNA for QRT-PCR analysis at day 3 of monolayer differentiation. ( F, G ) QRT-PCR analysis for marker genes of the three germ layers in control or TSA-treated ESCs (10 and 20 ng/ml) during EB differentiation. The EBs was treated by TSA from day 2 to 6 of EB differentiation. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: TSA induces early differentiation of ESCs and promotes mesodermal lineage differentiation. ( A ) Bright-field images, alkaline phosphatase staining of ESCs and representative immunofluorescence images of Oct4 staining in control or TSA-treated ESCs (10 and 20 ng/ml) in the presence of LIF. ( B ) Western blotting verification of H3, acetyl-H3, H4, and acetyl-H4 in control or TSA-treated ESCs (10 and 20 ng/ml). GAPDH was used as a loading control. ( C ) The relative expression levels of Oct4, Nanog, and Rex1 mRNA in control or TSA-treated ESCs (10 and 20 ng/ml). ( D, E ) QRT-PCR analysis for marker genes of three germ layers (endoderm, mesoderm and ectoderm) in control or TSA-treated ESCs (10 and 20 ng/ml), under the monolayer differentiation condition without LIF. The cells were treated by TSA after removing LIF for 24h and collected mRNA for QRT-PCR analysis at day 3 of monolayer differentiation. ( F, G ) QRT-PCR analysis for marker genes of the three germ layers in control or TSA-treated ESCs (10 and 20 ng/ml) during EB differentiation. The EBs was treated by TSA from day 2 to 6 of EB differentiation. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Staining, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Marker, Two Tailed Test

    Loss of HDAC1 or 3 enhances mesodermal lineage differentiation. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in shHDAC1 and shHDAC3 ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the knockdown of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis of mesoderm genes in shHDAC1 ESCs and control cells at the days 0, 3, 6, and 10 during EB differentiation. ( D ) QRT-PCR analysis of mesoderm genes in shHDAC3 ESCs and control cells during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, shHDAC1, and shHDAC3 cells after 9 days of EB formation. Green, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: Loss of HDAC1 or 3 enhances mesodermal lineage differentiation. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in shHDAC1 and shHDAC3 ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the knockdown of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis of mesoderm genes in shHDAC1 ESCs and control cells at the days 0, 3, 6, and 10 during EB differentiation. ( D ) QRT-PCR analysis of mesoderm genes in shHDAC3 ESCs and control cells during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, shHDAC1, and shHDAC3 cells after 9 days of EB formation. Green, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Staining, Western Blot, Quantitative RT-PCR, Immunofluorescence, Expressing, Two Tailed Test

    The histone deacetylase activity of HDACs is required for the regulation of mesoderm gene. ( A ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC1-OE (H1 OE), and TSA-treated H1-OE cells. GAPDH was used as a loading control. ( B, C ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H1-OE, and TSA-treated H1-OE cells. ( D ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC3-OE (H1 OE), and TSA-treated H3-OE cells. GAPDH was used as a loading control. ( E, F ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H3-OE, and TSA-treated H3-OE cells. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: The histone deacetylase activity of HDACs is required for the regulation of mesoderm gene. ( A ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC1-OE (H1 OE), and TSA-treated H1-OE cells. GAPDH was used as a loading control. ( B, C ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H1-OE, and TSA-treated H1-OE cells. ( D ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC3-OE (H1 OE), and TSA-treated H3-OE cells. GAPDH was used as a loading control. ( E, F ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H3-OE, and TSA-treated H3-OE cells. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Histone Deacetylase Assay, Activity Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test

    Ectopic expression of HDAC1 and 3 inhibits the differentiation into the mesodermal lineage in EBs. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in control, HDAC1-overexpression (HDAC1-OE), and HDAC3-overexpression (HDAC3-OE) ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the overexpression of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis for the mRNA levels of mesoderm genes in HDAC1-OE ESCs, HDAC3-OE ESCs and control cells during EB differentiation. ( D ) Western blotting analysis of the Gata4 and α-SMA protein levels in HDAC3-OE ESCs and control cell lines during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, HDAC1-OE, and HDAC3-OE cells after 9 days of EB formation. Red, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: Ectopic expression of HDAC1 and 3 inhibits the differentiation into the mesodermal lineage in EBs. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in control, HDAC1-overexpression (HDAC1-OE), and HDAC3-overexpression (HDAC3-OE) ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the overexpression of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis for the mRNA levels of mesoderm genes in HDAC1-OE ESCs, HDAC3-OE ESCs and control cells during EB differentiation. ( D ) Western blotting analysis of the Gata4 and α-SMA protein levels in HDAC3-OE ESCs and control cell lines during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, HDAC1-OE, and HDAC3-OE cells after 9 days of EB formation. Red, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Expressing, Staining, Over Expression, Western Blot, Quantitative RT-PCR, Immunofluorescence, Two Tailed Test

    9) Product Images from "Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma"

    Article Title: Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma

    Journal: Biomolecules

    doi: 10.3390/biom9100622

    shRNA-mediated knockdown of caveolin-1 expression of 1321N1 astrocytoma cells. ( A ) Immunoblot analysis of hHAP2Y 2 R, caveolin-1 (Cav-1), and GAPDH (control) expression in serum-starved wild-type (WT) 1321N1 cells (lane 1), human 1321N1 cells expressing hHAP2Y 2 R (hHAP2Y 2 R 1321N1 cells) (lane 3), or cells infected with Cav-1 shRNA lentiviral particles (lanes 2 and 4; Cav-1 knockdown (KD)). ( B ) Densitometric analysis of immunoblots indicates the level of Cav-1 normalized to GAPDH expression. Results are presented as the means ± S.E.M. (n = 3; *** p
    Figure Legend Snippet: shRNA-mediated knockdown of caveolin-1 expression of 1321N1 astrocytoma cells. ( A ) Immunoblot analysis of hHAP2Y 2 R, caveolin-1 (Cav-1), and GAPDH (control) expression in serum-starved wild-type (WT) 1321N1 cells (lane 1), human 1321N1 cells expressing hHAP2Y 2 R (hHAP2Y 2 R 1321N1 cells) (lane 3), or cells infected with Cav-1 shRNA lentiviral particles (lanes 2 and 4; Cav-1 knockdown (KD)). ( B ) Densitometric analysis of immunoblots indicates the level of Cav-1 normalized to GAPDH expression. Results are presented as the means ± S.E.M. (n = 3; *** p

    Techniques Used: shRNA, Expressing, Infection, Western Blot

    10) Product Images from "FoxP1 orchestration of ASD-relevant signaling pathways in the striatum"

    Article Title: FoxP1 orchestration of ASD-relevant signaling pathways in the striatum

    Journal: Genes & Development

    doi: 10.1101/gad.267989.115

    Regulation of ASD genes by Foxp1 in the mouse brain. ( A ) Representative immunoblot displaying reduced Foxp1 protein levels in the hippocampus (HIP) and striatum (STR), but not the neocortex (CTX), of Foxp1 +/− mice. Gapdh was used as a loading control. ( B ) Quantification of Foxp1 expression in adult Foxp1 +/− mouse brains. Data are represented as means ± SEM. n = 4 mice per genotype for each region. (*) P = 0.033 (hippocampus); (*) P = 0.0163 (striatum), Student's t -test, compared with wild-type levels normalized to Gapdh. ( C ) Venn diagram showing overlaps between the differentially expressed genes (DEGs) in the mouse and ASD gene lists (144 genes between the hippocampus and striatum [ P = 1.21 × 10 −26 ], 116 genes between the hippocampus and ASD [ P = 3.74 × 10 −9 ], and 43 genes between the striatum and ASD [ P = 0.002], hypergeometric test [ P -values were adjusted using Benjamini-Hochberg FDR procedure]). ( D ) Confirmation of salient ASD-related gene targets in independent striatal samples from Foxp1 +/− mice using quantitative RT–PCR (qRT–PCR). Data are represented as means ± SEM. n = 4 mice per genotype. With the exception of Dner , all qRT–PCR values displayed are significant at P
    Figure Legend Snippet: Regulation of ASD genes by Foxp1 in the mouse brain. ( A ) Representative immunoblot displaying reduced Foxp1 protein levels in the hippocampus (HIP) and striatum (STR), but not the neocortex (CTX), of Foxp1 +/− mice. Gapdh was used as a loading control. ( B ) Quantification of Foxp1 expression in adult Foxp1 +/− mouse brains. Data are represented as means ± SEM. n = 4 mice per genotype for each region. (*) P = 0.033 (hippocampus); (*) P = 0.0163 (striatum), Student's t -test, compared with wild-type levels normalized to Gapdh. ( C ) Venn diagram showing overlaps between the differentially expressed genes (DEGs) in the mouse and ASD gene lists (144 genes between the hippocampus and striatum [ P = 1.21 × 10 −26 ], 116 genes between the hippocampus and ASD [ P = 3.74 × 10 −9 ], and 43 genes between the striatum and ASD [ P = 0.002], hypergeometric test [ P -values were adjusted using Benjamini-Hochberg FDR procedure]). ( D ) Confirmation of salient ASD-related gene targets in independent striatal samples from Foxp1 +/− mice using quantitative RT–PCR (qRT–PCR). Data are represented as means ± SEM. n = 4 mice per genotype. With the exception of Dner , all qRT–PCR values displayed are significant at P

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR

    11) Product Images from "Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all"

    Article Title: Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19342

    5-Aza and the miR-708 inhibitor increased DKK3 expression and inhibited the Wnt signaling pathway in the B-ALL cell lines (A and B) . DKK3 mRNA and miR-708 expression were altered when the cells were treated with 5-aza and the miR-708 inhibitor. (C and D) Expression of β-catenin (cytoplasmic and nuclear), cyclin D1, GSK3β and p-GSK3β in the B-ALL cell lines after treatment with 5-aza and the miR-708 inhibitor, as determined by western blotting. The graphs show the corresponding band intensities of β-catenin, cyclin D1, GSK3β and p-GSK3β normalized to GAPDH and compared with the control. (E and F) . The images represent β-catenin (cytoplasmic and nuclear), cyclin D1 GSK3β and p- GSK3β expression in BALL-1 cells after treatment with 5-aza and the miR-708 inhibitor/mimics or negative controls (NC), as determined by western blotting. The data are presented as the means ± SDs of triplicate experiments. * P
    Figure Legend Snippet: 5-Aza and the miR-708 inhibitor increased DKK3 expression and inhibited the Wnt signaling pathway in the B-ALL cell lines (A and B) . DKK3 mRNA and miR-708 expression were altered when the cells were treated with 5-aza and the miR-708 inhibitor. (C and D) Expression of β-catenin (cytoplasmic and nuclear), cyclin D1, GSK3β and p-GSK3β in the B-ALL cell lines after treatment with 5-aza and the miR-708 inhibitor, as determined by western blotting. The graphs show the corresponding band intensities of β-catenin, cyclin D1, GSK3β and p-GSK3β normalized to GAPDH and compared with the control. (E and F) . The images represent β-catenin (cytoplasmic and nuclear), cyclin D1 GSK3β and p- GSK3β expression in BALL-1 cells after treatment with 5-aza and the miR-708 inhibitor/mimics or negative controls (NC), as determined by western blotting. The data are presented as the means ± SDs of triplicate experiments. * P

    Techniques Used: Expressing, Western Blot

    12) Product Images from "In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1? Axis"

    Article Title: In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1? Axis

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2011.04.011

    Effects of PGC-1α Overexpression in Surf1 −/− Mice (A) Expression analysis of PGC-1α . (Left panel) The PGC-1α transcript from muscle of each mouse genotype (three animals/genotype) was retrotranscribed into cDNA, normalized to that of the Hprt gene, and expressed as time-fold variations relative to the values obtained from wild-type (WT) animals. Solid blue, WT; blue outline, PGC-1α : MCK-PGC-1α transgenic mouse; solid red, Surf1 −/− , constitutive Surf1 knockout mouse; red outline, Surf1 −/− /PGC-1α : Surf1 −/− /MCK-PGC-1α double mutant mouse. Error bars represent the standard deviation (SD). (Right panel) Western blot immunovisualization of skeletal muscle proteins of the different genotypes, listed as above. Densitometric analysis of each band, normalized against that of GAPDH, revealed the following variations, relative to WT. (1) PGC-1α, 4.3 in MCK-PGC-1α ; 0.5 in Surf1 −/− ; 2.2 in Surf1 −/− /PGC-1α . (2) COX1, 1.8 in MCK-PGC-1α ; 0.2 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (3) COX5a, 5.0 in MCK-PGC-1α ; ∼0.0 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (B) MtDNA analysis in different genotypes (three animals/genotype). Color code is as in (A). MtDNA is expressed as number of DNA molecules per cell ( Cree et al., 2008 ). Error bars represent SD. (C) MRC activities in the different genotypes (three animals/genotype), expressed as nmoles/min/mg of protein. Note that the activity of cII has been multiplied by 10 for visualization clarity. Color code is as in (A). CS, citrate synthase; CI-IV, MRC complexes I–IV. Error bars represent the standard deviation (SD). The asterisks represent the significance levels calculated by unpaired, Student's two-tailed t test: ∗ p
    Figure Legend Snippet: Effects of PGC-1α Overexpression in Surf1 −/− Mice (A) Expression analysis of PGC-1α . (Left panel) The PGC-1α transcript from muscle of each mouse genotype (three animals/genotype) was retrotranscribed into cDNA, normalized to that of the Hprt gene, and expressed as time-fold variations relative to the values obtained from wild-type (WT) animals. Solid blue, WT; blue outline, PGC-1α : MCK-PGC-1α transgenic mouse; solid red, Surf1 −/− , constitutive Surf1 knockout mouse; red outline, Surf1 −/− /PGC-1α : Surf1 −/− /MCK-PGC-1α double mutant mouse. Error bars represent the standard deviation (SD). (Right panel) Western blot immunovisualization of skeletal muscle proteins of the different genotypes, listed as above. Densitometric analysis of each band, normalized against that of GAPDH, revealed the following variations, relative to WT. (1) PGC-1α, 4.3 in MCK-PGC-1α ; 0.5 in Surf1 −/− ; 2.2 in Surf1 −/− /PGC-1α . (2) COX1, 1.8 in MCK-PGC-1α ; 0.2 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (3) COX5a, 5.0 in MCK-PGC-1α ; ∼0.0 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (B) MtDNA analysis in different genotypes (three animals/genotype). Color code is as in (A). MtDNA is expressed as number of DNA molecules per cell ( Cree et al., 2008 ). Error bars represent SD. (C) MRC activities in the different genotypes (three animals/genotype), expressed as nmoles/min/mg of protein. Note that the activity of cII has been multiplied by 10 for visualization clarity. Color code is as in (A). CS, citrate synthase; CI-IV, MRC complexes I–IV. Error bars represent the standard deviation (SD). The asterisks represent the significance levels calculated by unpaired, Student's two-tailed t test: ∗ p

    Techniques Used: Pyrolysis Gas Chromatography, Over Expression, Mouse Assay, Expressing, Transgenic Assay, Knock-Out, Mutagenesis, Standard Deviation, Western Blot, Activity Assay, Two Tailed Test

    13) Product Images from "A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity"

    Article Title: A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity

    Journal: Nature immunology

    doi: 10.1038/ni.3393

    NS3 inhibits binding of RIG-I to 14-3-3ε, preventing the translocation of activated RIG-I to mitochondria ( a ) Ubiquitination of RIG-I-FLAG in transfected HEK293T cells that were mock-infected or infected with SeV (50 HAU/ml) for 19 h. WCLs were subjected to FLAG-PD, followed by IB with anti-ubiquitin (Ub) and anti-FLAG. ( b ) Ubiquitination of endogenous RIG-I in Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-Ub and anti-RIG-I. ( c ) Binding of endogenous RIG-I, TRIM25 and 14-3-3ε in transfected HEK293T cells that were infected with SeV (50 HAU/ml) for 23 h. WCLs were subjected to IP with anti-RIG-I (left) or anti-TRIM25 (right), followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. The data shown are from the same experiment. ( d ) Huh7 cells were mock-infected, or infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. ( e ) HEK293T cells were transfected with RIG-I-FLAG together with vector or increasing amounts of GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h, and FLAG-PD was performed. ( f ) Cytosol-mitochondria fractionation of WCLs from Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 22 h. IB was performed with anti-RIG-I, anti-MAVS and anti-GAPDH. RIG-I and NS3 expressions were determined in the WCL. Data are representative of at least 2 independent experiments ( a–f ).
    Figure Legend Snippet: NS3 inhibits binding of RIG-I to 14-3-3ε, preventing the translocation of activated RIG-I to mitochondria ( a ) Ubiquitination of RIG-I-FLAG in transfected HEK293T cells that were mock-infected or infected with SeV (50 HAU/ml) for 19 h. WCLs were subjected to FLAG-PD, followed by IB with anti-ubiquitin (Ub) and anti-FLAG. ( b ) Ubiquitination of endogenous RIG-I in Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-Ub and anti-RIG-I. ( c ) Binding of endogenous RIG-I, TRIM25 and 14-3-3ε in transfected HEK293T cells that were infected with SeV (50 HAU/ml) for 23 h. WCLs were subjected to IP with anti-RIG-I (left) or anti-TRIM25 (right), followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. The data shown are from the same experiment. ( d ) Huh7 cells were mock-infected, or infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. ( e ) HEK293T cells were transfected with RIG-I-FLAG together with vector or increasing amounts of GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h, and FLAG-PD was performed. ( f ) Cytosol-mitochondria fractionation of WCLs from Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 22 h. IB was performed with anti-RIG-I, anti-MAVS and anti-GAPDH. RIG-I and NS3 expressions were determined in the WCL. Data are representative of at least 2 independent experiments ( a–f ).

    Techniques Used: Binding Assay, Translocation Assay, Transfection, Infection, Plasmid Preparation, Fractionation

    14) Product Images from "Mild Functional Differences of Dynamin 2 Mutations Associated to Centronuclear Myopathy and Charcot-Marie-Tooth Peripheral Neuropathy"

    Article Title: Mild Functional Differences of Dynamin 2 Mutations Associated to Centronuclear Myopathy and Charcot-Marie-Tooth Peripheral Neuropathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027498

    MT dynamics in CNM patient fibroblasts. (A) MT dynamics in fibroblasts from control and CNM patients harboring the R465W or S619L mutation. Fibroblasts were left untreated or were treated with 5 µM nocodazole for 2 h at 37°C. To allow for partial recovery of MTs, nocodazole was removed and cells were placed at 37°C for 5 min. To view MTs, the cells were stained with β tubulin specific antibodies. Stabilized MTs were visualized with acetylated tubulin-specific antibodies. ( B ) Levels of acetylated tubulin are comparable for control and patient fibroblast lines. Equal amounts of indicated fibroblast lysates were subjected to SDS-PAGE and were analyzed by western blot employing anti-acetylated tubulin antibodies and controls (anti-EEA1, anti-dynamin 2 and anti-GAPDH).
    Figure Legend Snippet: MT dynamics in CNM patient fibroblasts. (A) MT dynamics in fibroblasts from control and CNM patients harboring the R465W or S619L mutation. Fibroblasts were left untreated or were treated with 5 µM nocodazole for 2 h at 37°C. To allow for partial recovery of MTs, nocodazole was removed and cells were placed at 37°C for 5 min. To view MTs, the cells were stained with β tubulin specific antibodies. Stabilized MTs were visualized with acetylated tubulin-specific antibodies. ( B ) Levels of acetylated tubulin are comparable for control and patient fibroblast lines. Equal amounts of indicated fibroblast lysates were subjected to SDS-PAGE and were analyzed by western blot employing anti-acetylated tubulin antibodies and controls (anti-EEA1, anti-dynamin 2 and anti-GAPDH).

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

    15) Product Images from "SUMO2/3 conjugation is an endogenous neuroprotective mechanism"

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

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1038/jcbfm.2011.112

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

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

    16) Product Images from ""

    Article Title:

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA118.000946

    Immunoblot validation of the abundance of differentially accumulating epididymosome proteins. A ,, Quantitative MS data were validated via immunoblotting of differentially accumulating proteins. Candidate proteins included representatives with the highest abundance (according to TMT reporter ion intensity) in epididymosomes from the proximal segment of the epididymis (caput) (ADAM7, B4GALT1, HSP90B1, MFGE8, PDIA6) in addition to proteins exhibiting increasing accumulation in cauda epididymosomes ( i.e. , ALDH2, CLU, PROM2, BAG6), and those that remained at relatively constant levels in epididymosomes sampled throughout the epididymis (PSMD7, DNM2, HSPA2). B ,, Corresponding MS quantification data are presented. C ,, Negative controls included sperm proteins of testicular origin (IZUMO1, ADAM3, ODF2), D ,, whereas positive controls included validated epididymosome/exosome proteins (GAPDH, FLOT1). Analyses were performed in triplicate using biological samples comprising pooled epididymosomes purified from 12 mice and representative immunoblots are depicted. E ,, A linear regression was performed to compare the quantification data obtained via TMT ( x , axis) and immunoblotting ( y , axis) analyses for each of the targeted epididymosome proteins, revealing significant correlation ( R , 2 = 0.61; p ,
    Figure Legend Snippet: Immunoblot validation of the abundance of differentially accumulating epididymosome proteins. A ,, Quantitative MS data were validated via immunoblotting of differentially accumulating proteins. Candidate proteins included representatives with the highest abundance (according to TMT reporter ion intensity) in epididymosomes from the proximal segment of the epididymis (caput) (ADAM7, B4GALT1, HSP90B1, MFGE8, PDIA6) in addition to proteins exhibiting increasing accumulation in cauda epididymosomes ( i.e. , ALDH2, CLU, PROM2, BAG6), and those that remained at relatively constant levels in epididymosomes sampled throughout the epididymis (PSMD7, DNM2, HSPA2). B ,, Corresponding MS quantification data are presented. C ,, Negative controls included sperm proteins of testicular origin (IZUMO1, ADAM3, ODF2), D ,, whereas positive controls included validated epididymosome/exosome proteins (GAPDH, FLOT1). Analyses were performed in triplicate using biological samples comprising pooled epididymosomes purified from 12 mice and representative immunoblots are depicted. E ,, A linear regression was performed to compare the quantification data obtained via TMT ( x , axis) and immunoblotting ( y , axis) analyses for each of the targeted epididymosome proteins, revealing significant correlation ( R , 2 = 0.61; p ,

    Techniques Used: Mass Spectrometry, Purification, Mouse Assay, Western Blot

    17) Product Images from "Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy"

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

    Journal: Nature communications

    doi: 10.1038/ncomms5425

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

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

    18) Product Images from "cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1"

    Article Title: cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

    Journal: Journal of Molecular and Cellular Cardiology

    doi: 10.1016/j.yjmcc.2014.11.012

    CCN1 promotes VSMC proliferation, migration and chemotaxis . Western blot analysis of CCN1 and GAPDH protein 24 h post-transfection with siNEG (□) or siCCN1(∎) (A). Relative BrDU incorporation 24–40 h post-transfection with siNEG of siCCN1; n = 3 (B). Barrier migration assay of siNEG (□) and siCCN1(∎) transfected cells after 18 h stimulation with 2.5 ng/ml or 10 ng/ml PDGF BB ; n = 6 (C and D). Boyden-chamber chemotaxis assay using 5 or 20 μg/ml recombinant CCN1 in the bottom chamber for 8 h; n = 6 (E and F). □ indicates controls; ∎ indicates stimulated cells. * indicates p
    Figure Legend Snippet: CCN1 promotes VSMC proliferation, migration and chemotaxis . Western blot analysis of CCN1 and GAPDH protein 24 h post-transfection with siNEG (□) or siCCN1(∎) (A). Relative BrDU incorporation 24–40 h post-transfection with siNEG of siCCN1; n = 3 (B). Barrier migration assay of siNEG (□) and siCCN1(∎) transfected cells after 18 h stimulation with 2.5 ng/ml or 10 ng/ml PDGF BB ; n = 6 (C and D). Boyden-chamber chemotaxis assay using 5 or 20 μg/ml recombinant CCN1 in the bottom chamber for 8 h; n = 6 (E and F). □ indicates controls; ∎ indicates stimulated cells. * indicates p

    Techniques Used: Migration, Chemotaxis Assay, Western Blot, Transfection, BrdU Incorporation Assay, Recombinant

    19) Product Images from "Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of ?1 integrin"

    Article Title: Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of ?1 integrin

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.078170

    4.1R is required for actin stress fiber and focal adhesion formation in keratinocytes. ( A ) Endogenous actin filament staining. Keratinocytes were cultured on fibronectin-coated surface for 36 hours, the cells were then fixed and stained for actin using Rhodamine–phalloidin. ( B ) Exogenously transfected GFP–actin. Keratinocytes were transiently transfected with GFP–actin. 24 hours after transformation, samples were fixed and viewed. All images shown were collected by wide-field epifluorescence microscopy, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). ( C ) Immunostaining of focal adhesion proteins. Cells were stained for β1 integrin, talin, paxillin and vinculin. All samples were counterstained for actin with Rhodamine–phalloidin (red) and DAPI (blue). Scale bars: 20 μm. ( D ) Localization of transiently transfected GFP–vinculin in keratinocytes. EGFP–vinculin was transiently transfected into 4.1R +/+ or 4.1R −/− primary keratinocytes using Fugene6. GFP-positive cells were sorted by FACS. Cells were viewed for fluorescence using a Zeiss Axiophot wide-field epifluorescence microscope. ( E ) Western blot analysis of actin and focal adhesion proteins. Total lysates (20 μg protein) were probed with indicated antibodies. GAPDH immunoblot is shown as a loading control. ( F ) Quantitative analysis from three independent experiments.
    Figure Legend Snippet: 4.1R is required for actin stress fiber and focal adhesion formation in keratinocytes. ( A ) Endogenous actin filament staining. Keratinocytes were cultured on fibronectin-coated surface for 36 hours, the cells were then fixed and stained for actin using Rhodamine–phalloidin. ( B ) Exogenously transfected GFP–actin. Keratinocytes were transiently transfected with GFP–actin. 24 hours after transformation, samples were fixed and viewed. All images shown were collected by wide-field epifluorescence microscopy, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). ( C ) Immunostaining of focal adhesion proteins. Cells were stained for β1 integrin, talin, paxillin and vinculin. All samples were counterstained for actin with Rhodamine–phalloidin (red) and DAPI (blue). Scale bars: 20 μm. ( D ) Localization of transiently transfected GFP–vinculin in keratinocytes. EGFP–vinculin was transiently transfected into 4.1R +/+ or 4.1R −/− primary keratinocytes using Fugene6. GFP-positive cells were sorted by FACS. Cells were viewed for fluorescence using a Zeiss Axiophot wide-field epifluorescence microscope. ( E ) Western blot analysis of actin and focal adhesion proteins. Total lysates (20 μg protein) were probed with indicated antibodies. GAPDH immunoblot is shown as a loading control. ( F ) Quantitative analysis from three independent experiments.

    Techniques Used: Staining, Cell Culture, Transfection, Transformation Assay, Epifluorescence Microscopy, Immunostaining, FACS, Fluorescence, Microscopy, Western Blot

    Expression and localization of 4.1R in keratinocytes. ( A ) Schematic view of 4.1R isoforms expressed in keratinocytes. The top diagram shows a general view of 4.1R exon organization, with alternatively spliced (black), constitutive (grey) and non-coding exons (open box) shown. Start codons are indicated. The lower diagrams show the exon composition of four isoforms revealed by sequencing of RT-PCR amplicons. ( B ) Immunoblot analysis of protein 4.1 members in keratinocytes. Total lysates (30 μg protein) were probed with polyclonal rabbit antibodies against 4.1R exon18 (a), 4.1R head piece (b), 4.1G head piece (c), 4.1N head piece (d) and 4.1B head piece (e). Quantitative analysis of immunoblot results from three independent experiments is shown in (f). GAPDH was used as loading control. ( C ) Confocal images of transfected GFP–4.1R. GFP–ATG2-4.1RΔexons14,15,17B was transiently expressed in live keratinocytes. A representative example of four images from a stack of 60 taken over a period of 10 hours, with a 10 minute interval between each panel is shown. Scale bar: 20 μm. ( D ) Immunofluorescence staining of endogenous 4.1R. Keratinocytes were cultured on fibronectin-coated surface for 36 hours, the cells were then fixed and stained using anti-4.1R-exon16 antibody.
    Figure Legend Snippet: Expression and localization of 4.1R in keratinocytes. ( A ) Schematic view of 4.1R isoforms expressed in keratinocytes. The top diagram shows a general view of 4.1R exon organization, with alternatively spliced (black), constitutive (grey) and non-coding exons (open box) shown. Start codons are indicated. The lower diagrams show the exon composition of four isoforms revealed by sequencing of RT-PCR amplicons. ( B ) Immunoblot analysis of protein 4.1 members in keratinocytes. Total lysates (30 μg protein) were probed with polyclonal rabbit antibodies against 4.1R exon18 (a), 4.1R head piece (b), 4.1G head piece (c), 4.1N head piece (d) and 4.1B head piece (e). Quantitative analysis of immunoblot results from three independent experiments is shown in (f). GAPDH was used as loading control. ( C ) Confocal images of transfected GFP–4.1R. GFP–ATG2-4.1RΔexons14,15,17B was transiently expressed in live keratinocytes. A representative example of four images from a stack of 60 taken over a period of 10 hours, with a 10 minute interval between each panel is shown. Scale bar: 20 μm. ( D ) Immunofluorescence staining of endogenous 4.1R. Keratinocytes were cultured on fibronectin-coated surface for 36 hours, the cells were then fixed and stained using anti-4.1R-exon16 antibody.

    Techniques Used: Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunofluorescence, Staining, Cell Culture

    20) Product Images from "Nek1 and TAZ Interact to Maintain Normal Levels of Polycystin 2"

    Article Title: Nek1 and TAZ Interact to Maintain Normal Levels of Polycystin 2

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2010090992

    Nek1 and TAZ form a negative feedback loop in the regulation of PC2 levels. (A) 293T cells were transfected with Myc-tagged Nek1 along with GST or GST-TAZ. Western blot was performed for GST, Myc, and GAPDH. (B) Immunoprecipitation was performed with
    Figure Legend Snippet: Nek1 and TAZ form a negative feedback loop in the regulation of PC2 levels. (A) 293T cells were transfected with Myc-tagged Nek1 along with GST or GST-TAZ. Western blot was performed for GST, Myc, and GAPDH. (B) Immunoprecipitation was performed with

    Techniques Used: Transfection, Western Blot, Immunoprecipitation

    21) Product Images from "GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3"

    Article Title: GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12839

    GATA4 trans-inactivates miR125b expression and indirectly regulates DKK3 expression A. Schematic illustration of 5 putative wild-type miR125b promoter constructs fused to a luciferase reporter gene using pGL-3 plasmid vector. B. Luciferase activity in extracts in Huh6 cells treated with or without GATA4 siRNA (Huh6 GATA4 siRNA) and transiently transfected with the 5 wild-type miR125b luciferase reporter constructs. Luciferase values are normalized to the Huh6 cells transfected with the empty pGL-3 basic vector. C. Schematic illustration of human wild-type or mutant miR125b-3 promotor constructs fused to a luciferase reporter gene. D. Luciferase activity in extracts from Huh6 cells treated with or without GATA4 shRNA cells and transiently transfected with the wild-type or mutant miR125b-3 luciferase reporter constructs. Average activity from five repeated samples was used to calculate luciferase activity. E. Schematic of the miR125b-3 upstream promoter containing a single GATA4-binding site. ChIP analyses revealed that GATA4 binds to the upstream GATA boxes in Huh6 cells; binding activity decreased in Huh6 cells treated with GATA4 siRNA. F. Western blot analysis of DKK3 and GATA4 expression in Huh6 cells treated with or without GATA4 siRNA cells; GAPDH served as the control. Huh6 NC: Huh6 cells treated with negative control miRNA. * P
    Figure Legend Snippet: GATA4 trans-inactivates miR125b expression and indirectly regulates DKK3 expression A. Schematic illustration of 5 putative wild-type miR125b promoter constructs fused to a luciferase reporter gene using pGL-3 plasmid vector. B. Luciferase activity in extracts in Huh6 cells treated with or without GATA4 siRNA (Huh6 GATA4 siRNA) and transiently transfected with the 5 wild-type miR125b luciferase reporter constructs. Luciferase values are normalized to the Huh6 cells transfected with the empty pGL-3 basic vector. C. Schematic illustration of human wild-type or mutant miR125b-3 promotor constructs fused to a luciferase reporter gene. D. Luciferase activity in extracts from Huh6 cells treated with or without GATA4 shRNA cells and transiently transfected with the wild-type or mutant miR125b-3 luciferase reporter constructs. Average activity from five repeated samples was used to calculate luciferase activity. E. Schematic of the miR125b-3 upstream promoter containing a single GATA4-binding site. ChIP analyses revealed that GATA4 binds to the upstream GATA boxes in Huh6 cells; binding activity decreased in Huh6 cells treated with GATA4 siRNA. F. Western blot analysis of DKK3 and GATA4 expression in Huh6 cells treated with or without GATA4 siRNA cells; GAPDH served as the control. Huh6 NC: Huh6 cells treated with negative control miRNA. * P

    Techniques Used: Expressing, Construct, Luciferase, Plasmid Preparation, Activity Assay, Transfection, Mutagenesis, shRNA, Binding Assay, Chromatin Immunoprecipitation, Western Blot, Negative Control

    22) Product Images from "Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase Cι to Associate with Microtubules and to Recruit Dynein"

    Article Title: Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase Cι to Associate with Microtubules and to Recruit Dynein

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807756200

    Rab2 requires GAPDH and PKC ι to associate with MTs. A , pure bovine brain tubulin (1 μg) was preassembled as outlined under “Experimental Procedures” and then incubated with Rab2 (200 ng), aPKCι (200 ng), or GAPDH
    Figure Legend Snippet: Rab2 requires GAPDH and PKC ι to associate with MTs. A , pure bovine brain tubulin (1 μg) was preassembled as outlined under “Experimental Procedures” and then incubated with Rab2 (200 ng), aPKCι (200 ng), or GAPDH

    Techniques Used: Incubation

    23) Product Images from "RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells"

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18851-4

    Effects of RGS7 mutation on RGS7 stability and activity. ( a ) The Human RGS7 protein, with conserved domains indicated as blocks, including the Dishevelled domain (DEP); G Protein Gamma-like domain (GGL); RGS domain (RGS). Somatic mutations indicated with arrows. Red triangles indicate deleterious mutations. ( b ) Three dimensional structure of RGS7 N-Terminus, predicting that R44 is involved in an H-bond network with S58 and D61. ( c ) Cells expressing wild-type or mutant RGS7 were treated with cycloheximide (CHX), collected at different time points and then immunoblotted with anti-FLAG antibody. Anti-Cyclin D1 was used as a control and anti-GAPDH was used for normalization. ( d ) A Schematic representation of the BRET-based assay to monitor G protein signaling cycle. Activation of the D2R causes the G protein heterotrimer to dissociate into Gα and Gβγ subunits. Released Gβγ subunits tagged with Venus fluorescent protein interacts with Nluc–tagged reporter G protein receptor kinase (GRK) to produce the BRET signal. Upon termination of D2R activation by antagonist haloperidol, Gαo subunit hydrolyses GTP and reassociates with Gβγ subunits, quenching the BRET signal. ( e ) Time course of normalized BRET responses recorded in a representative experiment. Left . The deactivation phase after antagonist application is shown. Wild-type RGS7 or mutant were transfected at equal amount of cDNA (210 ng) together with dopamine D2 receptor, Gαo, and BRET sensor pair. Right . Quantification of the exponential decay kinetics of the response. BRET values were averaged from four or six replicates. *P
    Figure Legend Snippet: Effects of RGS7 mutation on RGS7 stability and activity. ( a ) The Human RGS7 protein, with conserved domains indicated as blocks, including the Dishevelled domain (DEP); G Protein Gamma-like domain (GGL); RGS domain (RGS). Somatic mutations indicated with arrows. Red triangles indicate deleterious mutations. ( b ) Three dimensional structure of RGS7 N-Terminus, predicting that R44 is involved in an H-bond network with S58 and D61. ( c ) Cells expressing wild-type or mutant RGS7 were treated with cycloheximide (CHX), collected at different time points and then immunoblotted with anti-FLAG antibody. Anti-Cyclin D1 was used as a control and anti-GAPDH was used for normalization. ( d ) A Schematic representation of the BRET-based assay to monitor G protein signaling cycle. Activation of the D2R causes the G protein heterotrimer to dissociate into Gα and Gβγ subunits. Released Gβγ subunits tagged with Venus fluorescent protein interacts with Nluc–tagged reporter G protein receptor kinase (GRK) to produce the BRET signal. Upon termination of D2R activation by antagonist haloperidol, Gαo subunit hydrolyses GTP and reassociates with Gβγ subunits, quenching the BRET signal. ( e ) Time course of normalized BRET responses recorded in a representative experiment. Left . The deactivation phase after antagonist application is shown. Wild-type RGS7 or mutant were transfected at equal amount of cDNA (210 ng) together with dopamine D2 receptor, Gαo, and BRET sensor pair. Right . Quantification of the exponential decay kinetics of the response. BRET values were averaged from four or six replicates. *P

    Techniques Used: Mutagenesis, Activity Assay, Expressing, Bioluminescence Resonance Energy Transfer, Activation Assay, Transfection

    24) Product Images from "Twist1 and Twist2 contribute to cytokine downregulation following chronic NOD2 stimulation of human macrophages through the coordinated regulation of transcriptional repressors and activators"

    Article Title: Twist1 and Twist2 contribute to cytokine downregulation following chronic NOD2 stimulation of human macrophages through the coordinated regulation of transcriptional repressors and activators

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

    doi: 10.4049/jimmunol.1402808

    Upon chronic NOD2 stimulation, expression and cytokine promoter binding of the anti-inflammatory transcription factors c-Maf and Bmi1 are upregulated in a Twist-dependent manner (A) MDMs (n=8) were left untreated or pre-treated with 100μg/ml MDP for 48h, and then treated with 100μg/ml MDP for 4h (acute). Fold increase in c-Maf, Bmi1 and Akt1 mRNA was normalized to untreated MDMs + SEM. (B) MDMs (n=8) were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, Twist1 or Twist2 siRNA, alone or in combination, 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/ml MDP for 4h (acute) and assessed for c-Maf, Bmi1 and Akt1 mRNA expression. Fold mRNA expression normalized to untreated, scrambled siRNA-transfected MDMs + SEM. (C) MDMs were left untreated or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, or a combination of Twist1 and Twist2 siRNA ± vectors expressing c-Maf or Bmi1, and 24h later (total 48h MDP pre-treatment), MDMs were treated with 100μg/ml MDP for an additional 8h (acute) and c-Maf or Bmi1 expression was assessed by Western blot. GAPDH was assessed as a loading control. (D) MDMs were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled or a combination of Twist1 and Twist2 siRNA, and 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/ml MDP for an additional 4h (acute). Recruitment of c-Maf (n=4) and Bmi1 (n=5) to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA-transfected MDMs + SEM. (B-C) Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. *, p
    Figure Legend Snippet: Upon chronic NOD2 stimulation, expression and cytokine promoter binding of the anti-inflammatory transcription factors c-Maf and Bmi1 are upregulated in a Twist-dependent manner (A) MDMs (n=8) were left untreated or pre-treated with 100μg/ml MDP for 48h, and then treated with 100μg/ml MDP for 4h (acute). Fold increase in c-Maf, Bmi1 and Akt1 mRNA was normalized to untreated MDMs + SEM. (B) MDMs (n=8) were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, Twist1 or Twist2 siRNA, alone or in combination, 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/ml MDP for 4h (acute) and assessed for c-Maf, Bmi1 and Akt1 mRNA expression. Fold mRNA expression normalized to untreated, scrambled siRNA-transfected MDMs + SEM. (C) MDMs were left untreated or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, or a combination of Twist1 and Twist2 siRNA ± vectors expressing c-Maf or Bmi1, and 24h later (total 48h MDP pre-treatment), MDMs were treated with 100μg/ml MDP for an additional 8h (acute) and c-Maf or Bmi1 expression was assessed by Western blot. GAPDH was assessed as a loading control. (D) MDMs were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled or a combination of Twist1 and Twist2 siRNA, and 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/ml MDP for an additional 4h (acute). Recruitment of c-Maf (n=4) and Bmi1 (n=5) to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA-transfected MDMs + SEM. (B-C) Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. *, p

    Techniques Used: Expressing, Binding Assay, Transfection, Western Blot, Chromatin Immunoprecipitation

    25) Product Images from "The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma"

    Article Title: The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2258-x

    Characterization of apoptosis triggered by TCEA3 expression. a RH30 cells expressing TCEA3 or empty vector control were harvested for RNA. BAX and BCL2 gene expression were assayed by qRT-PCR. Scale bars 50 µm. b RH30 cells expressing TCEA3 or empty vector were assayed by western blot with antibodies against the indicated apoptotic marker proteins. GAPDH was used as a loading control. c RH30 cells expressing TCEA3 and empty vector were treated with caspase 8 inhibitor (Z-IETD-FMK, 40 μM), caspase 9 inhibitor (Z-LEHD-FMK, 40 μM) or a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 18 h. Immunofluorescence assay was done with anti TCEA3 antibody (green) and anti-cleaved caspase 3 antibody (red). DAPI (blue) was used to visualize nuclei. Scale bar is 50 µm. d Western blot assay on the same cells as in C. to confirm the protein expression of TCEA3, caspase 3 and tubulin as a loading control. * marks cleaved caspase 3. Error bars are S.E.M. Student t test; *** p
    Figure Legend Snippet: Characterization of apoptosis triggered by TCEA3 expression. a RH30 cells expressing TCEA3 or empty vector control were harvested for RNA. BAX and BCL2 gene expression were assayed by qRT-PCR. Scale bars 50 µm. b RH30 cells expressing TCEA3 or empty vector were assayed by western blot with antibodies against the indicated apoptotic marker proteins. GAPDH was used as a loading control. c RH30 cells expressing TCEA3 and empty vector were treated with caspase 8 inhibitor (Z-IETD-FMK, 40 μM), caspase 9 inhibitor (Z-LEHD-FMK, 40 μM) or a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 18 h. Immunofluorescence assay was done with anti TCEA3 antibody (green) and anti-cleaved caspase 3 antibody (red). DAPI (blue) was used to visualize nuclei. Scale bar is 50 µm. d Western blot assay on the same cells as in C. to confirm the protein expression of TCEA3, caspase 3 and tubulin as a loading control. * marks cleaved caspase 3. Error bars are S.E.M. Student t test; *** p

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Marker, Immunofluorescence

    26) Product Images from "Secreted Protein Acidic and Rich in Cysteine (SPARC) Enhances Cell Proliferation, Migration, and Epithelial Mesenchymal Transition, and SPARC Expression is Associated with Tumor Grade in Head and Neck Cancer"

    Article Title: Secreted Protein Acidic and Rich in Cysteine (SPARC) Enhances Cell Proliferation, Migration, and Epithelial Mesenchymal Transition, and SPARC Expression is Associated with Tumor Grade in Head and Neck Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18071556

    Investigation of SPARC treatment-induced signaling pathways by Western blot: Detroit 562 cells were treated with SPARC for 1.5 h and the protein extract was subjected to Western blot assay. ( A ) Phosphorylation status of ribosomal s6 kinase1 (RSK1), mitogen and stress-activated protein kinase (MSK) , and extracellular signal-regulated kinase (ERK); ( B ) phosphorylation status of p70S6K, AKT, 4e-BP1 and ( C ) investigation of the effect of ERK, RSK, and AKT inhibitor. Band intensity was normalized to GAPDH and values are expressed relative to the control group.
    Figure Legend Snippet: Investigation of SPARC treatment-induced signaling pathways by Western blot: Detroit 562 cells were treated with SPARC for 1.5 h and the protein extract was subjected to Western blot assay. ( A ) Phosphorylation status of ribosomal s6 kinase1 (RSK1), mitogen and stress-activated protein kinase (MSK) , and extracellular signal-regulated kinase (ERK); ( B ) phosphorylation status of p70S6K, AKT, 4e-BP1 and ( C ) investigation of the effect of ERK, RSK, and AKT inhibitor. Band intensity was normalized to GAPDH and values are expressed relative to the control group.

    Techniques Used: Western Blot

    27) Product Images from "Resveratrol sequentially induces replication and oxidative stresses to drive p53-CXCR2 mediated cellular senescence in cancer cells"

    Article Title: Resveratrol sequentially induces replication and oxidative stresses to drive p53-CXCR2 mediated cellular senescence in cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00315-4

    CXCR2 protects cells from undergoing stress-induced apoptosis. ( A ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured 3 days after treatment with RSV (25 μM) by flow cytometry. ( B ) Cells were treated as in ( A ) and whole-cell extracts were collected for Western blot analysis using BCL2 and BAX antibodies. ( C ) Downregulation of CXCR2 by siRNA as measured by RT-PCR and flow cytometry. U2OS cells were transfected with siRNA duplexes (200 nmol/L) specific to CXCR2 or scrabbled oligo in serum-free medium for 6 hours, then were incubated with complete medium for 24 h and then incubated with RSV for 3 days. ( D ) U2OS cells were treated the same as in ( C ) and apoptosis was measured by flow cytometry. ( E ) The U2OS cells were treated the same as in ( C ) and whole-cell extracts were collected for Western blot analysis using BCL2 antibodies. ( F ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured by flow cytometry 2 days after treatment with H 2 O 2 (400 μM). Results shown are representative of three independent experiments. ( G ) The shNeg and shCXCR2 NHF cells were treated the same as in ( F ) and apoptosis was measured by flow cytometry. The numbers shown below Western blot images are means (first row) and SE (second row) of band intensities relative to control. Signals on the immunoblots were analyzed by ImageJ, normalized with that of GAPDH.
    Figure Legend Snippet: CXCR2 protects cells from undergoing stress-induced apoptosis. ( A ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured 3 days after treatment with RSV (25 μM) by flow cytometry. ( B ) Cells were treated as in ( A ) and whole-cell extracts were collected for Western blot analysis using BCL2 and BAX antibodies. ( C ) Downregulation of CXCR2 by siRNA as measured by RT-PCR and flow cytometry. U2OS cells were transfected with siRNA duplexes (200 nmol/L) specific to CXCR2 or scrabbled oligo in serum-free medium for 6 hours, then were incubated with complete medium for 24 h and then incubated with RSV for 3 days. ( D ) U2OS cells were treated the same as in ( C ) and apoptosis was measured by flow cytometry. ( E ) The U2OS cells were treated the same as in ( C ) and whole-cell extracts were collected for Western blot analysis using BCL2 antibodies. ( F ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured by flow cytometry 2 days after treatment with H 2 O 2 (400 μM). Results shown are representative of three independent experiments. ( G ) The shNeg and shCXCR2 NHF cells were treated the same as in ( F ) and apoptosis was measured by flow cytometry. The numbers shown below Western blot images are means (first row) and SE (second row) of band intensities relative to control. Signals on the immunoblots were analyzed by ImageJ, normalized with that of GAPDH.

    Techniques Used: Flow Cytometry, Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Incubation

    28) Product Images from "ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers"

    Article Title: ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177649

    TGF-β signaling pathway is induced in gastrocnemius muscle from symptomatic hSOD1 G93A mice. (A) TGF-β (1, 2, 3), CTGF/CCN2 was detected by western-blot in protein extracts under non-reducing condition from wild-type (60 days old), pre-symptomatic (60 days old) hSOD1 G93A age-matched mice, wild-type (120 days old) and symptomatic (120 days old) hSOD1 G93A age-matched mice. GAPDH protein levels are shown as loading control. (B-C) Protein levels of TGF-β (1, 2, 3) and CTGF/CCN2 were quantified using densitometric analysis. Values correspond to the mean ± SEM of four animals for each experimental condition. One-way ANOVA, *** p
    Figure Legend Snippet: TGF-β signaling pathway is induced in gastrocnemius muscle from symptomatic hSOD1 G93A mice. (A) TGF-β (1, 2, 3), CTGF/CCN2 was detected by western-blot in protein extracts under non-reducing condition from wild-type (60 days old), pre-symptomatic (60 days old) hSOD1 G93A age-matched mice, wild-type (120 days old) and symptomatic (120 days old) hSOD1 G93A age-matched mice. GAPDH protein levels are shown as loading control. (B-C) Protein levels of TGF-β (1, 2, 3) and CTGF/CCN2 were quantified using densitometric analysis. Values correspond to the mean ± SEM of four animals for each experimental condition. One-way ANOVA, *** p

    Techniques Used: Mouse Assay, Western Blot

    29) Product Images from "A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency"

    Article Title: A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Journal: Scientific Reports

    doi: 10.1038/srep41408

    DMSA-SPIONs accumulate in endosomal compartments with increasing chase periods while aminolipid-SPIONs remain tethered at the cell surface. ( A ) Quantitative immunoblot analysis (equal protein loading) for the indicated organelle marker proteins in isolated fractions using DMSA-SPIONs with increasing chase periods of ( A ) 0 min, ( B ) 1 h, ( C ) 2 h, ( D ) 3 h, ( E ) 4 h or in fractions isolated using aminolipid-SPIONs ( F ) as a fold increase relative to total cell lysate (mean ± SEM, n = 3). Na + K + (Na + K + -ATPase) is a PM-localized integral membrane protein, EEA1 marks early endosomes, Rab7 late endosomes and Lamp1 lysosomes. RIB (Ribophorin) and GAPDH represent endoplasmic reticulum and cytosol, respectively. TCL - total cell lysate; UB - Unbound/non-magnetic fraction and B - Bound/magnetic fraction. ( G ) Silver staining of total cell lysate (TCL), bound/magnetic fraction isolated using SPIONs functionalized with DMSA (LYS) or with aminolipids (PM). The distinct protein profile in the bound fraction as observed by the lane scan underscores the enrichment of specific protein subsets (M - Marker, SeeBlue plus2 rainbow protein marker (Invitrogen)). TEM analysis of the fractions isolated using DMSA- ( H ) and aminolipid- ( I ) coated SPIONs. Scale bar = 0.5 μm.
    Figure Legend Snippet: DMSA-SPIONs accumulate in endosomal compartments with increasing chase periods while aminolipid-SPIONs remain tethered at the cell surface. ( A ) Quantitative immunoblot analysis (equal protein loading) for the indicated organelle marker proteins in isolated fractions using DMSA-SPIONs with increasing chase periods of ( A ) 0 min, ( B ) 1 h, ( C ) 2 h, ( D ) 3 h, ( E ) 4 h or in fractions isolated using aminolipid-SPIONs ( F ) as a fold increase relative to total cell lysate (mean ± SEM, n = 3). Na + K + (Na + K + -ATPase) is a PM-localized integral membrane protein, EEA1 marks early endosomes, Rab7 late endosomes and Lamp1 lysosomes. RIB (Ribophorin) and GAPDH represent endoplasmic reticulum and cytosol, respectively. TCL - total cell lysate; UB - Unbound/non-magnetic fraction and B - Bound/magnetic fraction. ( G ) Silver staining of total cell lysate (TCL), bound/magnetic fraction isolated using SPIONs functionalized with DMSA (LYS) or with aminolipids (PM). The distinct protein profile in the bound fraction as observed by the lane scan underscores the enrichment of specific protein subsets (M - Marker, SeeBlue plus2 rainbow protein marker (Invitrogen)). TEM analysis of the fractions isolated using DMSA- ( H ) and aminolipid- ( I ) coated SPIONs. Scale bar = 0.5 μm.

    Techniques Used: Marker, Isolation, Silver Staining, Transmission Electron Microscopy

    30) Product Images from "Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma"

    Article Title: Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11299

    Expression of Kv1.3 potassium channel in different pancreatic ductal adenocarcinoma cell lines A . Histogram of the distribution of Kcna3 gene expression in a panel of pancreatic cancer cell lines from Affymetrix U133 Gene Chip. Arbitrary intensity units of the probe ID (207237_at) for Kcna3; the intensity values were normalized with dCHIP2006 software ( www.dchip.org ) B . The relative level of gene expression as determined by quantitative RT-PCR was calculated using qBase_Biogazelle software, which allows a multiple reference genes-normalization and performs inter-run calibration. Actb and Ywhaz of non-tumoral HPDE cells were set as reference genes (value 1) to normalize the gene expression. C . Whole cell extracts (50 μg/lane) from different PDAC cell lines were loaded on SDS-PAGE. Western blot revealed Kv1.3 bands (multiple bands are presumably due to glycosylation according to manufacturer or to degradation products) at around 65 kDa. These bands correlated with Kv1.3 expression since none of the bands were present in a cell line silenced for Kv1.3 (not shown). The same blot was developed with the antibody against GAPDH (45 kDa) as loading control. D . Whole cell extract (Tot ext), enriched membranous fraction (Memb) and Percoll-purified mitochondria (mitos) fractions obtained from Colo357 were loaded at equal protein concentration (40 μg/lane). Enrichment in the mitochondrial marker Bak and decrease of the intensity of SERCA (ER marker) and PMCA (PM marker) indicates a higher purity of the mitochondrial preparation. Results in C-D are representative of Western blots from three independent experiments.
    Figure Legend Snippet: Expression of Kv1.3 potassium channel in different pancreatic ductal adenocarcinoma cell lines A . Histogram of the distribution of Kcna3 gene expression in a panel of pancreatic cancer cell lines from Affymetrix U133 Gene Chip. Arbitrary intensity units of the probe ID (207237_at) for Kcna3; the intensity values were normalized with dCHIP2006 software ( www.dchip.org ) B . The relative level of gene expression as determined by quantitative RT-PCR was calculated using qBase_Biogazelle software, which allows a multiple reference genes-normalization and performs inter-run calibration. Actb and Ywhaz of non-tumoral HPDE cells were set as reference genes (value 1) to normalize the gene expression. C . Whole cell extracts (50 μg/lane) from different PDAC cell lines were loaded on SDS-PAGE. Western blot revealed Kv1.3 bands (multiple bands are presumably due to glycosylation according to manufacturer or to degradation products) at around 65 kDa. These bands correlated with Kv1.3 expression since none of the bands were present in a cell line silenced for Kv1.3 (not shown). The same blot was developed with the antibody against GAPDH (45 kDa) as loading control. D . Whole cell extract (Tot ext), enriched membranous fraction (Memb) and Percoll-purified mitochondria (mitos) fractions obtained from Colo357 were loaded at equal protein concentration (40 μg/lane). Enrichment in the mitochondrial marker Bak and decrease of the intensity of SERCA (ER marker) and PMCA (PM marker) indicates a higher purity of the mitochondrial preparation. Results in C-D are representative of Western blots from three independent experiments.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Software, Quantitative RT-PCR, SDS Page, Western Blot, Purification, Protein Concentration, Marker

    31) Product Images from "Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells"

    Article Title: Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094259

    The effect of ASN on the protein level and gene expression for GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The total level of GSK-3β was determined using Western blot analysis. ( A ) Immunoreactivity of GSK-3β and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of GSK-3β immunoreactivity. Results were normalized to GAPDH levels. ( C ) The gene expression for total GSK-3β was measured with real-time PCR. Data represent the mean value ± S.E.M. for 5 independent experiments. * p
    Figure Legend Snippet: The effect of ASN on the protein level and gene expression for GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The total level of GSK-3β was determined using Western blot analysis. ( A ) Immunoreactivity of GSK-3β and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of GSK-3β immunoreactivity. Results were normalized to GAPDH levels. ( C ) The gene expression for total GSK-3β was measured with real-time PCR. Data represent the mean value ± S.E.M. for 5 independent experiments. * p

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

    The effect of CDK5 and GSK-3β inhibitors on ASN-evoked increase in Tau phosphorylation. PC12 cells were incubated with 10 μM ASN in the presence of 10 μM inhibitors for 48 h. BML-259 and SB-216763 were used as CDK5 and GSK-3β inhibitor, respectively. The level of Tau phosphorylation at Ser396 was determined using the Western blotting method. ( A ) Immunoreactivity of p-Tau (Ser396) and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of p-Tau (Ser396) immunoreactivity. Results were normalized to GAPDH levels. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p
    Figure Legend Snippet: The effect of CDK5 and GSK-3β inhibitors on ASN-evoked increase in Tau phosphorylation. PC12 cells were incubated with 10 μM ASN in the presence of 10 μM inhibitors for 48 h. BML-259 and SB-216763 were used as CDK5 and GSK-3β inhibitor, respectively. The level of Tau phosphorylation at Ser396 was determined using the Western blotting method. ( A ) Immunoreactivity of p-Tau (Ser396) and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of p-Tau (Ser396) immunoreactivity. Results were normalized to GAPDH levels. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p

    Techniques Used: Incubation, Western Blot

    The effect of ASN on the phosphorylation state of GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The phosphorylation status of GSK-3β at Ser9 and Tyr216 was determined using Western blot analysis. ( A ) Immunoreactivity of p-GSK-3β (Ser9). ( B ) Immunoreactivity of p-GSK-3β (Tyr216). GAPDH was used as a loading control. Data represent the mean value ± S.E.M. for 5 independent experiments. * p
    Figure Legend Snippet: The effect of ASN on the phosphorylation state of GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The phosphorylation status of GSK-3β at Ser9 and Tyr216 was determined using Western blot analysis. ( A ) Immunoreactivity of p-GSK-3β (Ser9). ( B ) Immunoreactivity of p-GSK-3β (Tyr216). GAPDH was used as a loading control. Data represent the mean value ± S.E.M. for 5 independent experiments. * p

    Techniques Used: Incubation, Western Blot

    32) Product Images from "DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA"

    Article Title: DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1299-x

    DAP5 positively regulates cap-independent translation of DSCR1.4 by binding to 5′UTR of DSCR1.4. a , b DAP5 interacts with hDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated hDSCR1.4 5′UTR constructs were incubated with SHSY5Y cell extracts. The region interacting with DAP5 was confirmed by Western blot. GAPDH and hDSCR1.4 were used as negative control. Nonbiotinylated hDSCR1.4 UTR was used as competitor. c DAP5 binds to proximal 136 nucleotides of mDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated mDSCR1.4 5′UTR transcripts were incubated with N2A cell extracts. Binding region was verified by Western blot. d , e A reduction of DAP5 decreases cap-independent translation efficiency of DSCR1.4 5′UTR. d SHSY5Y and e N2A cells were transfected with control siRNA (siCon) or DAP5 siRNA (siDAP5) and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and siCon transfected cells was set as 1. The bars represent the mean ± SEM ( n = 7, n = 7). f , g DAP5 overexpression upregulated cap-independent translation of DSCR1.4 5′UTR. f SHSY5Y and g N2A cells were transfected with Flag Mock or Flag DAP5 plasmids and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and Flag Mock transfected cells was set as 1. The bars represent the mean ± SEM ( n = 5, n = 5). h , i DAP5 deficiency results in reduction of DSCR1.4 protein levels. siCon or siDAP5 was transfected on h SHSY5Y and i N2A cells. DAP5 knockdown was confirmed by Western blot using anti-DAP5. j , k Overexpressed DAP5 increases DSCR1.4 protein levels. j SHSY5Y and k N2A cells were transfected with Flag Mock or Flag DAP5 and incubated for 24 h. DAP5 overexpression was verified by Western blot using anti-Flag antibody. l The increase of DSCR1.4 expressions by DAP5 overexpression results from cap-independent translation. SHSY5Ycells were transfected with Flag Mock or Flag DAP5 and 18 h later incubated and followed by 200 μM rapamycin treatment for 6 h. The rapamycin activity was analyzed by phosphorylation status of S6RP protein. DAP5 overexpression was confirmed by anti-Flag antibody. Data information: In d – g , * P
    Figure Legend Snippet: DAP5 positively regulates cap-independent translation of DSCR1.4 by binding to 5′UTR of DSCR1.4. a , b DAP5 interacts with hDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated hDSCR1.4 5′UTR constructs were incubated with SHSY5Y cell extracts. The region interacting with DAP5 was confirmed by Western blot. GAPDH and hDSCR1.4 were used as negative control. Nonbiotinylated hDSCR1.4 UTR was used as competitor. c DAP5 binds to proximal 136 nucleotides of mDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated mDSCR1.4 5′UTR transcripts were incubated with N2A cell extracts. Binding region was verified by Western blot. d , e A reduction of DAP5 decreases cap-independent translation efficiency of DSCR1.4 5′UTR. d SHSY5Y and e N2A cells were transfected with control siRNA (siCon) or DAP5 siRNA (siDAP5) and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and siCon transfected cells was set as 1. The bars represent the mean ± SEM ( n = 7, n = 7). f , g DAP5 overexpression upregulated cap-independent translation of DSCR1.4 5′UTR. f SHSY5Y and g N2A cells were transfected with Flag Mock or Flag DAP5 plasmids and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and Flag Mock transfected cells was set as 1. The bars represent the mean ± SEM ( n = 5, n = 5). h , i DAP5 deficiency results in reduction of DSCR1.4 protein levels. siCon or siDAP5 was transfected on h SHSY5Y and i N2A cells. DAP5 knockdown was confirmed by Western blot using anti-DAP5. j , k Overexpressed DAP5 increases DSCR1.4 protein levels. j SHSY5Y and k N2A cells were transfected with Flag Mock or Flag DAP5 and incubated for 24 h. DAP5 overexpression was verified by Western blot using anti-Flag antibody. l The increase of DSCR1.4 expressions by DAP5 overexpression results from cap-independent translation. SHSY5Ycells were transfected with Flag Mock or Flag DAP5 and 18 h later incubated and followed by 200 μM rapamycin treatment for 6 h. The rapamycin activity was analyzed by phosphorylation status of S6RP protein. DAP5 overexpression was confirmed by anti-Flag antibody. Data information: In d – g , * P

    Techniques Used: Binding Assay, In Vitro, Construct, Incubation, Western Blot, Negative Control, Transfection, Luciferase, Activity Assay, Over Expression

    BDNF makes cap-independent translation of DSCR1.4 mRNA more actively by increasing DAP5 expression. a , b BDNF treatment on DIV 3 hippocampal neuron increases protein levels of DAP5 and DSCR1.4 but not DSCR1.4 mRNA level. Vehicle (DDW) or 30 μM BDNF were treated for 1 h. a The protein levels were confirmed by Western blot. GAPDH and phosphorylation of ERK were used as a loading control and marker of BDNF activity, respectively. b Endogenous DSCR1.4 mRNA levels were analyzed by qRT-PCR and were normalized to β-actin. The bars represent the mean ± SEM ( n = 3). c Cap-independent translation is essential for DSCR1.4 protein accumulation by BDNF. DIV 3 hippocampal neurons were treated with vehicle (DMSO), 100 μM RAD001(RAD) or 50 mg/ml cycloheximide (CHX) for 3 h followed by BDNF treatment for 1 h. The levels of each protein were confirmed by Western blot. The numbers at the bottom indicate the fold relative to a vehicle. The amount of DSCR1.4 was normalized to GAPDH. d , e BDNF raises cap-independent translation activity of DSCR1.4 mRNA. At 24 h after d pRF hDSCR1.4 5′UTR or e pRF mDSCR1.4 5′UTR vectors were transfected into DIV 2 hippocampal neurons, Vehicle (DDW) and BDNF were treated to the neurons for 1 h. The bars represent the mean ± SEM ( e ; n = 5, F; n = 3). f BDNF increases the interaction between DAP5 and DSCR1.4 5′UTR. In vitro transcribed biotin-DSCR1.4 5′UTR was incubated with extracts of the vehicle (DDW) or 30 μM BDNF-treated DIV 3 mouse hippocampal neurons. DAP5 binding was measured by Western blot. Phospho-ERK was used to confirm the activity of BDNF. GADPH was used as a loading control and negative control. g , h BDNF increases the cap-independent local translation of DSCR1.4 mRNA in axon as well as soma. EGFP and pRF mDSCR1.4 5′ 3′ UTR vectors were co-transfected into DIV 2 mouse hippocampal neurons. At 24 h later, 100 μM anisomycin was treated for 3 h and then 30 μM BDNF was treated for 1 h, followed by 5 μM puromycin treatment for 40 min. To detect newly synthesized FLUC proteins, Puro-PLA assay was conducted. g Representative image obtained from confocal microscopy. h The graph shows relative fluorescence intensity measured by Image J. The bars represent the mean ± SEM (Vehicle; n = 11, Anisomycin; n = 12, BDNF; n = 11, BDNF + Anisomycin; n = 11). Scale bar, 30 μm. Data information: In d , e , h , * P
    Figure Legend Snippet: BDNF makes cap-independent translation of DSCR1.4 mRNA more actively by increasing DAP5 expression. a , b BDNF treatment on DIV 3 hippocampal neuron increases protein levels of DAP5 and DSCR1.4 but not DSCR1.4 mRNA level. Vehicle (DDW) or 30 μM BDNF were treated for 1 h. a The protein levels were confirmed by Western blot. GAPDH and phosphorylation of ERK were used as a loading control and marker of BDNF activity, respectively. b Endogenous DSCR1.4 mRNA levels were analyzed by qRT-PCR and were normalized to β-actin. The bars represent the mean ± SEM ( n = 3). c Cap-independent translation is essential for DSCR1.4 protein accumulation by BDNF. DIV 3 hippocampal neurons were treated with vehicle (DMSO), 100 μM RAD001(RAD) or 50 mg/ml cycloheximide (CHX) for 3 h followed by BDNF treatment for 1 h. The levels of each protein were confirmed by Western blot. The numbers at the bottom indicate the fold relative to a vehicle. The amount of DSCR1.4 was normalized to GAPDH. d , e BDNF raises cap-independent translation activity of DSCR1.4 mRNA. At 24 h after d pRF hDSCR1.4 5′UTR or e pRF mDSCR1.4 5′UTR vectors were transfected into DIV 2 hippocampal neurons, Vehicle (DDW) and BDNF were treated to the neurons for 1 h. The bars represent the mean ± SEM ( e ; n = 5, F; n = 3). f BDNF increases the interaction between DAP5 and DSCR1.4 5′UTR. In vitro transcribed biotin-DSCR1.4 5′UTR was incubated with extracts of the vehicle (DDW) or 30 μM BDNF-treated DIV 3 mouse hippocampal neurons. DAP5 binding was measured by Western blot. Phospho-ERK was used to confirm the activity of BDNF. GADPH was used as a loading control and negative control. g , h BDNF increases the cap-independent local translation of DSCR1.4 mRNA in axon as well as soma. EGFP and pRF mDSCR1.4 5′ 3′ UTR vectors were co-transfected into DIV 2 mouse hippocampal neurons. At 24 h later, 100 μM anisomycin was treated for 3 h and then 30 μM BDNF was treated for 1 h, followed by 5 μM puromycin treatment for 40 min. To detect newly synthesized FLUC proteins, Puro-PLA assay was conducted. g Representative image obtained from confocal microscopy. h The graph shows relative fluorescence intensity measured by Image J. The bars represent the mean ± SEM (Vehicle; n = 11, Anisomycin; n = 12, BDNF; n = 11, BDNF + Anisomycin; n = 11). Scale bar, 30 μm. Data information: In d , e , h , * P

    Techniques Used: Expressing, Western Blot, Marker, Activity Assay, Quantitative RT-PCR, Transfection, In Vitro, Incubation, Binding Assay, Negative Control, Synthesized, Proximity Ligation Assay, Confocal Microscopy, Fluorescence

    hDSCR1.4 and mDSCR1.4 mRNA are cap-independently translated and have cis-regulatory elements in their 5′UTRs. a Cap-independent translational regulation contributes to DSCR1.4 protein expression. SHSY5Y cells were treated with DMSO or 200 μM rapamycin or 50 mg/ml cycloheximide for the indicated times. The levels of endogenous proteins were measured by Western blotting (WB) using anti-DSCR1.4, anti-phosphoS6RP, anti-GAPDH antibodies. GAPDH was used as a loading control. The activity of rapamycin was analyzed by the phosphorylation status of S6RP. The numbers at the bottom indicate the fold increases relative to control. The amount of DSCR1.4 was normalized to GAPDH. b Schematic representation of pRF bicistronic luciferase plasmids used for observing cap-independent translation activity of human and mouse DSCR1.4 5′UTR. c , d hDSCR1.4 5′UTR and mDSCR1.4 5′UTR induce cap-independent translation initiation. c SHSY5Y and d N2A cells were transfected with the bicistronic reporter plasmids and were incubated for 24 h. pRF β-globin was used as a negative control. pRF EMCV and pRF p53 were used as the positive control. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 4). e , f hDSCR1.4 5′UTR and mDSCR1.4 5′UTR prefer cap-independent translation to cap-dependent translation. e SHSY5Y cells were transfected with in vitro transcribed m7G capped or ApppG capped hDSCR1.4 5′UTR-FLUC. f N2A cells were transfected with in vitro transcribed m7G capped or ApppG capped mDSCR1.4 5′UTR-FLUC. Transfected cells were incubated for 6 h and were harvested. Translation activity is shown as the ratio of FLUC to FLUC mRNA. Translation activity of m7G capped transcripts was set as 1. The bars represent the mean ± SEM ( n = 4, n = 3). g , i The 5′ proximal 119 nucleotides sequence of hDSCR1.4 5′UTR is important for cap-independent translation activity of hDSCR1.4 5′UTR. pRF plasmids with serial deletion constructs and six nucleotides mutant construct were transfected into g SHSY5Y cells and i mouse primary hippocampal neurons. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3). h , j The 5′ proximal 136 nucleotides are essential for cap-independent translation activity of mDSCR1.4 5′UTR. Indicated pRF plasmids were transfected into h N2A cells and j mouse hippocampal neurons. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 3). Data information: In c – j , * P
    Figure Legend Snippet: hDSCR1.4 and mDSCR1.4 mRNA are cap-independently translated and have cis-regulatory elements in their 5′UTRs. a Cap-independent translational regulation contributes to DSCR1.4 protein expression. SHSY5Y cells were treated with DMSO or 200 μM rapamycin or 50 mg/ml cycloheximide for the indicated times. The levels of endogenous proteins were measured by Western blotting (WB) using anti-DSCR1.4, anti-phosphoS6RP, anti-GAPDH antibodies. GAPDH was used as a loading control. The activity of rapamycin was analyzed by the phosphorylation status of S6RP. The numbers at the bottom indicate the fold increases relative to control. The amount of DSCR1.4 was normalized to GAPDH. b Schematic representation of pRF bicistronic luciferase plasmids used for observing cap-independent translation activity of human and mouse DSCR1.4 5′UTR. c , d hDSCR1.4 5′UTR and mDSCR1.4 5′UTR induce cap-independent translation initiation. c SHSY5Y and d N2A cells were transfected with the bicistronic reporter plasmids and were incubated for 24 h. pRF β-globin was used as a negative control. pRF EMCV and pRF p53 were used as the positive control. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 4). e , f hDSCR1.4 5′UTR and mDSCR1.4 5′UTR prefer cap-independent translation to cap-dependent translation. e SHSY5Y cells were transfected with in vitro transcribed m7G capped or ApppG capped hDSCR1.4 5′UTR-FLUC. f N2A cells were transfected with in vitro transcribed m7G capped or ApppG capped mDSCR1.4 5′UTR-FLUC. Transfected cells were incubated for 6 h and were harvested. Translation activity is shown as the ratio of FLUC to FLUC mRNA. Translation activity of m7G capped transcripts was set as 1. The bars represent the mean ± SEM ( n = 4, n = 3). g , i The 5′ proximal 119 nucleotides sequence of hDSCR1.4 5′UTR is important for cap-independent translation activity of hDSCR1.4 5′UTR. pRF plasmids with serial deletion constructs and six nucleotides mutant construct were transfected into g SHSY5Y cells and i mouse primary hippocampal neurons. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3). h , j The 5′ proximal 136 nucleotides are essential for cap-independent translation activity of mDSCR1.4 5′UTR. Indicated pRF plasmids were transfected into h N2A cells and j mouse hippocampal neurons. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 3). Data information: In c – j , * P

    Techniques Used: Expressing, Western Blot, Activity Assay, Luciferase, Transfection, Incubation, Negative Control, Positive Control, Plasmid Preparation, In Vitro, Sequencing, Construct, Mutagenesis

    33) Product Images from "MicroRNA-93 Regulates Hypoxia-Induced Autophagy by Targeting ULK1"

    Article Title: MicroRNA-93 Regulates Hypoxia-Induced Autophagy by Targeting ULK1

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/2709053

    The knockdown of ULK1 by siRNA suppressed hypoxia-induced autophagy. (a, b) MEFs and CHO cells were transfected with three pairs of siRNAs to ULK for 24 h, respectively, si-ULK1 (488), si-ULK1 (2457), and si-ULK1 (3310), setting NC as a control. Cell lysates were prepared and subjected to Western blot analysis by using anti-ULK1 and anti-Gapdh. The densitometric ratios from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (c) Si-ULK1 (488) was transfected into MEFs for 12 h, followed by hypoxic treatment or normoxic treatment for another 24 h. Cell lysates were analyzed by Western blot using anti-ULK1, anti-P62, anti-LC3, and anti-Gapdh. Densitometric ratios of the samples were quantified by using ImageJ. (d) MEFs were treated as the same as (c). Cells were fixed and immunostained by anti-ULK1 (green) and anti-P62 (red) antibodies. Scale bar, 20 μ m.
    Figure Legend Snippet: The knockdown of ULK1 by siRNA suppressed hypoxia-induced autophagy. (a, b) MEFs and CHO cells were transfected with three pairs of siRNAs to ULK for 24 h, respectively, si-ULK1 (488), si-ULK1 (2457), and si-ULK1 (3310), setting NC as a control. Cell lysates were prepared and subjected to Western blot analysis by using anti-ULK1 and anti-Gapdh. The densitometric ratios from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (c) Si-ULK1 (488) was transfected into MEFs for 12 h, followed by hypoxic treatment or normoxic treatment for another 24 h. Cell lysates were analyzed by Western blot using anti-ULK1, anti-P62, anti-LC3, and anti-Gapdh. Densitometric ratios of the samples were quantified by using ImageJ. (d) MEFs were treated as the same as (c). Cells were fixed and immunostained by anti-ULK1 (green) and anti-P62 (red) antibodies. Scale bar, 20 μ m.

    Techniques Used: Transfection, Western Blot

    34) Product Images from "Preclinical efficacy for a novel tyrosine kinase inhibitor, ArQule 531 against acute myeloid leukemia"

    Article Title: Preclinical efficacy for a novel tyrosine kinase inhibitor, ArQule 531 against acute myeloid leukemia

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-019-0821-7

    ARQ 531 modulates AML pro-survival kinases. Immunoblot analysis for MOLM-13, MV4-11, and OCI-AML3 AML cell lines (representative of 2-3 independent blots). All cell lines were serum starved overnight followed by 10-min treatment of five to 10 million cells with DMSO, increasing concentrations of ARQ 531 in comparison with 50 nM of quizartinib (Quiz.) or 50 nM gilteritinib (Gilt.), 0.1 μM midostaurin (Mido.), 0.5 μM dasatinib (Das.), 1 μM entospletinib (Ento.), or a combination of 1 μM ARQ 531 and Ento. Twenty to 25 μg of total protein lysate was loaded per lane. GAPDH, HSP90, or β-Actin were used as loading controls. a, b Modulation of phosphorylated SFK and downstream targets in FLT3-ITD cell lines MOLM-13 and MV-11, respectively. c , d Modulation of phosphorylated SYK and BTK in in all three cell lines
    Figure Legend Snippet: ARQ 531 modulates AML pro-survival kinases. Immunoblot analysis for MOLM-13, MV4-11, and OCI-AML3 AML cell lines (representative of 2-3 independent blots). All cell lines were serum starved overnight followed by 10-min treatment of five to 10 million cells with DMSO, increasing concentrations of ARQ 531 in comparison with 50 nM of quizartinib (Quiz.) or 50 nM gilteritinib (Gilt.), 0.1 μM midostaurin (Mido.), 0.5 μM dasatinib (Das.), 1 μM entospletinib (Ento.), or a combination of 1 μM ARQ 531 and Ento. Twenty to 25 μg of total protein lysate was loaded per lane. GAPDH, HSP90, or β-Actin were used as loading controls. a, b Modulation of phosphorylated SFK and downstream targets in FLT3-ITD cell lines MOLM-13 and MV-11, respectively. c , d Modulation of phosphorylated SYK and BTK in in all three cell lines

    Techniques Used:

    35) Product Images from "Development of an oncolytic Herpes Simplex Virus using a tumor-specific HIF-responsive promoter"

    Article Title: Development of an oncolytic Herpes Simplex Virus using a tumor-specific HIF-responsive promoter

    Journal: Cancer gene therapy

    doi: 10.1038/cgt.2010.62

    ICP4 (34kDa) and thymidine kinase (TK) expression by HIF-E6L-HSV and HIF-V6R-HSV E5 cells were infected at MOI 0.25 with the indicated (a) HIF-E6L-HSV (clones 1-5) or (b) HIF-V6R-HSV (clones 7, 9, 16, 18, 21, 8) and then grown under normoxia. As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 30μg of total cell lysate was examined by immunoblotting for the presence of full length ICP4 (175 kDa), truncated ICP4 (34kDa), TK (40 kDa), and GAPDH (loading control, 36 kDa) protein expression. The ICP4 bands are shown at two different exposures to better visualize the 175 kDa and 34 kDa ICP4 proteins. Asterisks indicate a non-specific band that is cross-reactive with the anti-ICP4 antibody.
    Figure Legend Snippet: ICP4 (34kDa) and thymidine kinase (TK) expression by HIF-E6L-HSV and HIF-V6R-HSV E5 cells were infected at MOI 0.25 with the indicated (a) HIF-E6L-HSV (clones 1-5) or (b) HIF-V6R-HSV (clones 7, 9, 16, 18, 21, 8) and then grown under normoxia. As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 30μg of total cell lysate was examined by immunoblotting for the presence of full length ICP4 (175 kDa), truncated ICP4 (34kDa), TK (40 kDa), and GAPDH (loading control, 36 kDa) protein expression. The ICP4 bands are shown at two different exposures to better visualize the 175 kDa and 34 kDa ICP4 proteins. Asterisks indicate a non-specific band that is cross-reactive with the anti-ICP4 antibody.

    Techniques Used: Expressing, Infection

    Strains HIF-E6L-HSV and HIF-V6R-HSV express full length ICP4 (175 kDa) and expression is not HIF-dependent LN229 cells were infected at MOI 0.05 with the indicated HIF-E6L-HSV (clones 1-5) or HIF-V6R-HSV (clones 6-22) clone and then grown under normoxia (N) or hypoxia (H). As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 20μg of total cell lysate was western blotted for full length ICP4 (175 kDa) protein expression. GAPDH (36 kDa) or actin (42 kDa) protein expression was used as a loading control. M=molecular weight standard lane.
    Figure Legend Snippet: Strains HIF-E6L-HSV and HIF-V6R-HSV express full length ICP4 (175 kDa) and expression is not HIF-dependent LN229 cells were infected at MOI 0.05 with the indicated HIF-E6L-HSV (clones 1-5) or HIF-V6R-HSV (clones 6-22) clone and then grown under normoxia (N) or hypoxia (H). As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 20μg of total cell lysate was western blotted for full length ICP4 (175 kDa) protein expression. GAPDH (36 kDa) or actin (42 kDa) protein expression was used as a loading control. M=molecular weight standard lane.

    Techniques Used: Expressing, Infection, Western Blot, Molecular Weight

    36) Product Images from "Human Cytomegalovirus-Induced Degradation of CYTIP Modulates Dendritic Cell Adhesion and Migration"

    Article Title: Human Cytomegalovirus-Induced Degradation of CYTIP Modulates Dendritic Cell Adhesion and Migration

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00461

    Human cytomegalovirus (HCMV) infection of mature dendritic cells (mDCs) leads to decreased CYTIP expression levels . (A) mDCs were either mock- or herpes simplex virus type 1 (HSV-1)-infected and harvested 24 hpi, (B) mock- or HCMV-infected and sorted based on their green fluorescence protein expression 24 hpi. (A,B) Protein lysates were prepared and subjected to Western blot analyses with antibodies specific for CYTIP, cytohesin-1, ICP0, IE2, or GAPDH. One representative experiment out of at least three is shown. (C) Quantification of CYTIP and cytohesin-1 protein expression levels in HSV-1-infected or HCMV-positive mDCs, summarizing at least three independent experiments. Expression of CYTIP and cytohesin-1 was normalized to the GAPDH control. Mock was set to 100%. Significant changes (**** = p
    Figure Legend Snippet: Human cytomegalovirus (HCMV) infection of mature dendritic cells (mDCs) leads to decreased CYTIP expression levels . (A) mDCs were either mock- or herpes simplex virus type 1 (HSV-1)-infected and harvested 24 hpi, (B) mock- or HCMV-infected and sorted based on their green fluorescence protein expression 24 hpi. (A,B) Protein lysates were prepared and subjected to Western blot analyses with antibodies specific for CYTIP, cytohesin-1, ICP0, IE2, or GAPDH. One representative experiment out of at least three is shown. (C) Quantification of CYTIP and cytohesin-1 protein expression levels in HSV-1-infected or HCMV-positive mDCs, summarizing at least three independent experiments. Expression of CYTIP and cytohesin-1 was normalized to the GAPDH control. Mock was set to 100%. Significant changes (**** = p

    Techniques Used: Infection, Expressing, Fluorescence, Western Blot

    37) Product Images from "The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles"

    Article Title: The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/7094781

    Western blot analysis of SOD1, SOD2, SOD3, catalase, and TRX in HUVECs (a–e) and MVs (g). GSH (total content) was analysed using a specific kit (f); the left panel shows the standard curve and the right panel the total GSH content. Y: young; S: senescent. HUVEC protein data were normalized against β -actin (a–d) or GAPDH (e); however, the GSH data (f) were normalized against the protein content of the sample. Protein data for the MVs was normalized against the intensity of red Ponceau staining. Bars represent mean ± SD ( n = 3 pools). ∗ p
    Figure Legend Snippet: Western blot analysis of SOD1, SOD2, SOD3, catalase, and TRX in HUVECs (a–e) and MVs (g). GSH (total content) was analysed using a specific kit (f); the left panel shows the standard curve and the right panel the total GSH content. Y: young; S: senescent. HUVEC protein data were normalized against β -actin (a–d) or GAPDH (e); however, the GSH data (f) were normalized against the protein content of the sample. Protein data for the MVs was normalized against the intensity of red Ponceau staining. Bars represent mean ± SD ( n = 3 pools). ∗ p

    Techniques Used: Western Blot, Staining

    38) Product Images from "Helicobacter pylori-induced Sonic Hedgehog expression is regulated by NFκB pathway activation: The use of a novel in vitro model to study epithelial response to infection"

    Article Title: Helicobacter pylori-induced Sonic Hedgehog expression is regulated by NFκB pathway activation: The use of a novel in vitro model to study epithelial response to infection

    Journal: Helicobacter

    doi: 10.1111/hel.12152

    H. pylori -induced NFκB activation and Shh expression (A) NFκB status was measured using protein lysates extracted from organoids infected with H. pylori for 0, 2, 4, and 24 hours. Changes in IκBα, IKKα and GAPDH expression were measured by western blot. Quantification of Changes in IκBα relative to GAPDH is shown in B . Data are expressed as the mean + SEM, where *P
    Figure Legend Snippet: H. pylori -induced NFκB activation and Shh expression (A) NFκB status was measured using protein lysates extracted from organoids infected with H. pylori for 0, 2, 4, and 24 hours. Changes in IκBα, IKKα and GAPDH expression were measured by western blot. Quantification of Changes in IκBα relative to GAPDH is shown in B . Data are expressed as the mean + SEM, where *P

    Techniques Used: Activation Assay, Expressing, Infection, Western Blot

    39) Product Images from "Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells"

    Article Title: Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells

    Journal: British Journal of Cancer

    doi: 10.1038/s41416-018-0284-6

    Analysis of predicted upstream regulators via ingenuity pathway analysis and biochemical confirmation. a Scatter plot depicting the predicted activation/inhibition status of upstream regulators belonging to the category of ‘transcription factors’. The x -axis represents the activation z -score, and the y -axis represents the negative logarithm of the significance of overlap between a regulator’s target genes and the differentially expressed genes in Pfn KD vs control samples. Regulators with an overlap p -value ≤0.01, and z -scores ≥2.0 or ≤−2.0 are considered to be significantly activated or inhibited, respectively. b List of target genes for the transcription factors SMAD3 and SPDEF, and their fold-change of expression (hatched bar: direction of gene changes consistent with predicted activation of SMAD3; grey bar: direction of gene changes not consistent; white bar: effects of direction of gene changes unknown). c Immunoblot analyses of SMAD3 and pSMAD3C from 3D BME-MoT extracts of MDA-231 cells transfected with the indicated siRNAs (GAPDH blot—loading control). d Immunoblot analyses of FAK, pFAK, SMAD3 and pSMAD3C from 3D extracts of control- vs Pfn1-shRNA expressing MDA-231 cells cultured in BME with or without collagen-I (data representative from three independent experiments)
    Figure Legend Snippet: Analysis of predicted upstream regulators via ingenuity pathway analysis and biochemical confirmation. a Scatter plot depicting the predicted activation/inhibition status of upstream regulators belonging to the category of ‘transcription factors’. The x -axis represents the activation z -score, and the y -axis represents the negative logarithm of the significance of overlap between a regulator’s target genes and the differentially expressed genes in Pfn KD vs control samples. Regulators with an overlap p -value ≤0.01, and z -scores ≥2.0 or ≤−2.0 are considered to be significantly activated or inhibited, respectively. b List of target genes for the transcription factors SMAD3 and SPDEF, and their fold-change of expression (hatched bar: direction of gene changes consistent with predicted activation of SMAD3; grey bar: direction of gene changes not consistent; white bar: effects of direction of gene changes unknown). c Immunoblot analyses of SMAD3 and pSMAD3C from 3D BME-MoT extracts of MDA-231 cells transfected with the indicated siRNAs (GAPDH blot—loading control). d Immunoblot analyses of FAK, pFAK, SMAD3 and pSMAD3C from 3D extracts of control- vs Pfn1-shRNA expressing MDA-231 cells cultured in BME with or without collagen-I (data representative from three independent experiments)

    Techniques Used: Activation Assay, Inhibition, Expressing, Multiple Displacement Amplification, Transfection, shRNA, Cell Culture

    Effects of perturbation of SMAD3 on 3D outgrowth of MDA-231 cells in BME matrix. a – b a: SMAD3 and GAPDH (loading control) immunoblots of MDA-231 cells transfected with either empty vector (EV) control or SMAD3 overexpression vector (cells were transfected with 1 µg plasmid in 35 mm culture dish). Note that the SMAD3 immunoblot image was acquired at a very low exposure to avoid signal saturation of the overexpression band. b Summarises the outgrowth of SMAD3 overexpression group relative to EV control group of cells in BME matrix. c – d c : SMAD3 and GAPDH (loading control) immunoblots of total cell extracts of control and Pfn1-shRNA expressing MDA-231 cells transiently transfected with the indicated siRNAs. d Summarises the outgrowth of the various transfected groups relative to the control group (control shRNA expressers transfected with control siRNA) in BME matrix. Gene silencing and overexpression-based outgrowth data summarised from two independent experiments, with three technical replicates per experiment). * p
    Figure Legend Snippet: Effects of perturbation of SMAD3 on 3D outgrowth of MDA-231 cells in BME matrix. a – b a: SMAD3 and GAPDH (loading control) immunoblots of MDA-231 cells transfected with either empty vector (EV) control or SMAD3 overexpression vector (cells were transfected with 1 µg plasmid in 35 mm culture dish). Note that the SMAD3 immunoblot image was acquired at a very low exposure to avoid signal saturation of the overexpression band. b Summarises the outgrowth of SMAD3 overexpression group relative to EV control group of cells in BME matrix. c – d c : SMAD3 and GAPDH (loading control) immunoblots of total cell extracts of control and Pfn1-shRNA expressing MDA-231 cells transiently transfected with the indicated siRNAs. d Summarises the outgrowth of the various transfected groups relative to the control group (control shRNA expressers transfected with control siRNA) in BME matrix. Gene silencing and overexpression-based outgrowth data summarised from two independent experiments, with three technical replicates per experiment). * p

    Techniques Used: Multiple Displacement Amplification, Western Blot, Transfection, Plasmid Preparation, Over Expression, shRNA, Expressing

    Effect of Pfn1 depletion on single cell outgrowth of BCC in 3D BME matrix. a Pfn1 and Pfn2 immunoblot analyses of total cell extracts of MDA-231 cells stably expressing either control or Pfn1-shRNA (GAPDH blot serves as a loading control). b Representative images of outgrowth of control and Pfn1-shRNA MDA-231 cells on 3D BME matrix from an initial seeding of 500 cells/well in a 384-well plate. c A bar graph summarizing the final number of cells (counted on day 10) in control vs Pfn1 shRNA groups for different seeding densities. All data are normalised to the final number of cells in control group for initial seeding density equal to 250 cells/well based on cell-count analyses of 3 technical replicates/condition from two independent experiments. d-g ; d , f Immunoblot analyses of Pfn1 and Pfn2 from total cell extracts of MDA-231 ( d ) and MDA-157 ( f ) cells following transient transfection with the indicated siRNAs (tubulin blot serves as a loading control). The bar graphs in e and g summarise the outgrowth of Pfn1 and Pfn2 KD MDA-231 and MDA-157 cells, respectively, on 3D BME matrix 7 days after seeding relative to the respective control siRNA transfected cell lines (data summarised from two independent experiments with three technical replicates per experiment) * p
    Figure Legend Snippet: Effect of Pfn1 depletion on single cell outgrowth of BCC in 3D BME matrix. a Pfn1 and Pfn2 immunoblot analyses of total cell extracts of MDA-231 cells stably expressing either control or Pfn1-shRNA (GAPDH blot serves as a loading control). b Representative images of outgrowth of control and Pfn1-shRNA MDA-231 cells on 3D BME matrix from an initial seeding of 500 cells/well in a 384-well plate. c A bar graph summarizing the final number of cells (counted on day 10) in control vs Pfn1 shRNA groups for different seeding densities. All data are normalised to the final number of cells in control group for initial seeding density equal to 250 cells/well based on cell-count analyses of 3 technical replicates/condition from two independent experiments. d-g ; d , f Immunoblot analyses of Pfn1 and Pfn2 from total cell extracts of MDA-231 ( d ) and MDA-157 ( f ) cells following transient transfection with the indicated siRNAs (tubulin blot serves as a loading control). The bar graphs in e and g summarise the outgrowth of Pfn1 and Pfn2 KD MDA-231 and MDA-157 cells, respectively, on 3D BME matrix 7 days after seeding relative to the respective control siRNA transfected cell lines (data summarised from two independent experiments with three technical replicates per experiment) * p

    Techniques Used: Multiple Displacement Amplification, Stable Transfection, Expressing, shRNA, Cell Counting, Transfection

    40) Product Images from "A Critical Role of Autophagy in Regulating Microglia Polarization in Neurodegeneration"

    Article Title: A Critical Role of Autophagy in Regulating Microglia Polarization in Neurodegeneration

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2018.00378

    TNF-α disrupted the autophagic flux in microglia. (A) Dose-dependent effect of the lysosome inhibitor BafA1 treatment for 2 h on LC3II accumulation in BV2 cells, as evaluated by western blotting. N = 3. (B) Effect of TNF-α on LC3II levels during lysosome inhibition. BV2 cells were treated with 5 ng/ml TNF-α for 24 h, and then added with BafA1(50 nM) for 2 h before subjected to western blotting. Actin served as loading controls in panels A,B . N = 4. (C,D) Autophagic assay in microglia treated with TNF-α, BafA1, or in combination. BV2 cells were transfected with RFP-GFP-tandem fluorescent LC3 cDNA for 24 h before treatment. Confocal microscope pictures showing yellow (GFP and RFP overlap) and red LC3 puncta formation in different groups. Scale bar, 10 μm. LC3 dots were visualized and quantified from at least 30 cells per group. (E) Effect of TNF-α on lysosomal biogenesis. BV2 cells were treated with 5 ng/ml TNF-α for 3, 12, or 24 h. The cytosolic and nuclear fractions were subjected to western blotting analysis of TFEB, with GAPDH and Histone 2B as the cytosolic and nuclear loading controls, respectively. (F,G) Effect of TNF-α on lysosomal protein LAMP1 (F) and LAMP2 (G) levels in BV2 cells. N = 3. ∗ P
    Figure Legend Snippet: TNF-α disrupted the autophagic flux in microglia. (A) Dose-dependent effect of the lysosome inhibitor BafA1 treatment for 2 h on LC3II accumulation in BV2 cells, as evaluated by western blotting. N = 3. (B) Effect of TNF-α on LC3II levels during lysosome inhibition. BV2 cells were treated with 5 ng/ml TNF-α for 24 h, and then added with BafA1(50 nM) for 2 h before subjected to western blotting. Actin served as loading controls in panels A,B . N = 4. (C,D) Autophagic assay in microglia treated with TNF-α, BafA1, or in combination. BV2 cells were transfected with RFP-GFP-tandem fluorescent LC3 cDNA for 24 h before treatment. Confocal microscope pictures showing yellow (GFP and RFP overlap) and red LC3 puncta formation in different groups. Scale bar, 10 μm. LC3 dots were visualized and quantified from at least 30 cells per group. (E) Effect of TNF-α on lysosomal biogenesis. BV2 cells were treated with 5 ng/ml TNF-α for 3, 12, or 24 h. The cytosolic and nuclear fractions were subjected to western blotting analysis of TFEB, with GAPDH and Histone 2B as the cytosolic and nuclear loading controls, respectively. (F,G) Effect of TNF-α on lysosomal protein LAMP1 (F) and LAMP2 (G) levels in BV2 cells. N = 3. ∗ P

    Techniques Used: Western Blot, Inhibition, Transfection, Microscopy

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    Western Blot:

    Article Title: Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers
    Article Snippet: .. The following antibodies were used for western blotting: anti-AP2A1/adaptin (1:1,000; BD Biosciences, 610502), anti-ACTB/β actin (1:20,000; Sigma-Aldrich, A5441), anti-LC3B (1:1,000; Cell Signaling Technology, 2775), anti-ATG7 (1:1,000; Cell Signaling Technology, 8558), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738), anti-GAPDH (1:1,000; Millipore, MAB374), anti-ATG5 (1:1000; Cosmo Bio, TMD-PH-AT5), anti-YAP1 (1:1,000; Cell Signaling Technology, 4912) and anti-phospho-YAP1 (1:1,000; Cell Signaling Technology, 4911). .. For immunofluorescence studies, the anti-LC3 was from NanoTools (1:50; NanoTools 0231–100/LC3–5F10).

    Article Title: Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice
    Article Snippet: .. Western blot analysis was done with Abr antiserum (1∶200) and Bcr (N-20)(1∶500, Santa Cruz) or GAPDH antibodies (1∶5000, Millipore). .. ELISA The IL-6 levels in the serum and lungs of mice and PASMCs supernatants were measured by Enzyme-Linked Immunosorbent Assay (ELISA) according to the manufacturer's instructions (eBioscience).

    Incubation:

    Article Title: Regulation of the p19Arf/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice
    Article Snippet: .. The membranes were blocked 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline supplied with 0.1% Tween 20 (TBS-T) and incubated with rabbit anti-p53 (1:500, Novocastra), ac-p53 (1:1000, Cell Signaling), ac-H3 (1:10 000, Sigma), p16 (1:200, Santa Cruz) or pp53 (S18) (1:500, RyD), rat anti-p19 (1:1000, Abcam), or mouse anti-Gapdh (1:1500, Millipore) antibodies in blocking buffer, o/n at 4 °C. .. After extensive washing with TBS-T, membranes were incubated for 1 h at RT with horseradish peroxidase-conjugated goat antibodies to mouse (1:2000, Dako), rabbit (1:5000, Amersham), or rat (1:1000, Amersham) IgGs in blocking buffer.

    Article Title: Long QT syndrome caveolin‐3 mutations differentially modulate Kv4 and Cav1.2 channels to contribute to action potential prolongation
    Article Snippet: .. To detect multiple proteins, blots were cut and incubated with anti‐Cav3 (catalogue number 610421; Becton‐Dickinson Biosciences, Franklin Lakes, NJ, USA) or anti‐GAPDH (MAB374; Millipore Sigma, Burlington, MA, USA) and then incubated with HRP‐conjugated secondary antibodies. .. Electrophysiological experiments were carried out in the whole‐cell configuration of the patch clamp technique at room temperature using the Axopatch 200B amplifier with pCLAMP, version 10.2 (Axon Instruments, Foster City, CA, USA).

    other:

    Article Title: Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts
    Article Snippet: The followings are the reagents used in this study and their sources: PDGF-BB, LPA, LMB, FITC-conjugated goat antimouse secondary antibody, and mouse anti-β-actin monoclonal antibody from Sigma-Aldrich (St. Louis, MO); DMEM, FBS, penicillin, and streptomycin from Gibco/BRL Life Technologies, Inc (Carlsbad, CA); PI3K inhibitor LY 294002, anti-rabbit monoclonal antibodies against phospho-AMPKα (Thr172 ) and GSK-3β (Ser9 ), anti-mouse monoclonal antibodies against phospho-Bad (Ser136 ) and phospho-Akt (Ser473 ), and anti-rabbit polyclonal antibodies against AMPK-α, Akt, and GSK-3β from Cell Signaling Technology (Denver, MA); compound C, AICAR, SH-5, L-NAME, and L-NMMA from Calbiochem (San Diego, CA); mouse anti-GAPDH monoclonal antibody from Chemicon (Bedford, MA); anti-rabbit monoclonal antibodies against ACC1 and phospho-ACC1 (Ser79 ) from Upstate (Waltham, MA); horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies from Vector Laboratories (Burlingame, CA); Lipofectamine™ RNAiMAX and mounting solution including DAPI from Invitrogen Life Technologies (Carlsbad, CA); protein assay kit from Bio-Rad Laboratories (Hercules, CA); enhanced chemiluminescence (ECL) system and Amersham hyperfilm™ ECL from GE Healthcare (Buckinghamshire, UK); sense and complement strands of human AMPK-α1 and α2 siRNAs, si CONTROL complete kit, and 5 × siRNA buffer from Dharmacon Inc. (Lafayette, CO); 5'-ITC from Biomol Research Labs (Plymouth Meeting, PA).

    Blocking Assay:

    Article Title: Regulation of the p19Arf/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice
    Article Snippet: .. The membranes were blocked 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline supplied with 0.1% Tween 20 (TBS-T) and incubated with rabbit anti-p53 (1:500, Novocastra), ac-p53 (1:1000, Cell Signaling), ac-H3 (1:10 000, Sigma), p16 (1:200, Santa Cruz) or pp53 (S18) (1:500, RyD), rat anti-p19 (1:1000, Abcam), or mouse anti-Gapdh (1:1500, Millipore) antibodies in blocking buffer, o/n at 4 °C. .. After extensive washing with TBS-T, membranes were incubated for 1 h at RT with horseradish peroxidase-conjugated goat antibodies to mouse (1:2000, Dako), rabbit (1:5000, Amersham), or rat (1:1000, Amersham) IgGs in blocking buffer.

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    Millipore anti gapdh
    Expression of <t>Cav3‐WT,</t> ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with <t>GAPDH</t> loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.
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    Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.

    Journal: The Journal of Physiology

    Article Title: Long QT syndrome caveolin‐3 mutations differentially modulate Kv4 and Cav1.2 channels to contribute to action potential prolongation

    doi: 10.1113/JP276014

    Figure Lengend Snippet: Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.

    Article Snippet: To detect multiple proteins, blots were cut and incubated with anti‐Cav3 (catalogue number 610421; Becton‐Dickinson Biosciences, Franklin Lakes, NJ, USA) or anti‐GAPDH (MAB374; Millipore Sigma, Burlington, MA, USA) and then incubated with HRP‐conjugated secondary antibodies.

    Techniques: Expressing, Transfection, Confocal Microscopy, Western Blot, Plasmid Preparation, Negative Control

    For figure legend, see page 2133. Figure 5 ( See previous page ). The transcription co-activator YAP1 is a key player in the autophagy-dependent proliferation and invasion of TN BC cells. ( A ) Western blots showing phosphorylated-YAP1 (P-YAP1), YAP1, and AP2A1 proteins in stable cell lines following 3D culture. AP2A1 is used as internal control for protein loading. The bar graph (right panel) shows the corresponding quantification of P-YAP1/YAP1 protein level ratios. ( B ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3D culture. GAPDH is used as an internal control for total mRNA expression. ( C ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with 3-methyladenine (3-MA; 20 mM) for 3 h. ( D ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3-MA treatment. GAPDH is used as an internal control for total mRNA expression. ( E ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with rapamycin (2 μM) for 3 h. ( F ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following rapamycin treatment. GAPDH is used as an internal control for total mRNA expression. ( G ) Left, western blots showing YAP1 protein levels in MDA231 cells after transfection with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. AP2A1 is used as an internal control for protein loading. Middle, representative bright field images from Control- or YAP1-depleted cells cultured in 3D, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Right, the bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( H ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from MDA231 cells transiently transfected with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. for 72 h prior to the assay. Left histogram: Numbers of invading cells, which passed through a Transwell over 6 h of incubation. Right panel: Percentage of cells, relative to siCtrl (100%), which passed through a Transwell over 6 h of incubation. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( I ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from autophagy-deficient MDA231 cells (36 h of transfection with si ATG7 or si ATG5 , as indicated), transfected again (36 h) with an empty vector or a vector expressing YAP1-S127A, a nonphosphorylable mutant form of YAP1. Numbers of invading cells, which passed through a Transwell over 6 h of incubation, are shown. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( J ) Results are from autophagy-proficient (sh Ctrl ) or autophagy-deficient (sh ATG5 ) MDA231 cells, transfected with an empty vector or a vector expressing YAP1-S127A, after 3 d of 3D culture. Representative bright field images from ATG5-depleted cells expressing or not YAP1-S127A, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 5 independent experiments). P -values are based on the Student t test.

    Journal: Autophagy

    Article Title: Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers

    doi: 10.4161/15548627.2014.981788

    Figure Lengend Snippet: For figure legend, see page 2133. Figure 5 ( See previous page ). The transcription co-activator YAP1 is a key player in the autophagy-dependent proliferation and invasion of TN BC cells. ( A ) Western blots showing phosphorylated-YAP1 (P-YAP1), YAP1, and AP2A1 proteins in stable cell lines following 3D culture. AP2A1 is used as internal control for protein loading. The bar graph (right panel) shows the corresponding quantification of P-YAP1/YAP1 protein level ratios. ( B ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3D culture. GAPDH is used as an internal control for total mRNA expression. ( C ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with 3-methyladenine (3-MA; 20 mM) for 3 h. ( D ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3-MA treatment. GAPDH is used as an internal control for total mRNA expression. ( E ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with rapamycin (2 μM) for 3 h. ( F ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following rapamycin treatment. GAPDH is used as an internal control for total mRNA expression. ( G ) Left, western blots showing YAP1 protein levels in MDA231 cells after transfection with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. AP2A1 is used as an internal control for protein loading. Middle, representative bright field images from Control- or YAP1-depleted cells cultured in 3D, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Right, the bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( H ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from MDA231 cells transiently transfected with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. for 72 h prior to the assay. Left histogram: Numbers of invading cells, which passed through a Transwell over 6 h of incubation. Right panel: Percentage of cells, relative to siCtrl (100%), which passed through a Transwell over 6 h of incubation. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( I ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from autophagy-deficient MDA231 cells (36 h of transfection with si ATG7 or si ATG5 , as indicated), transfected again (36 h) with an empty vector or a vector expressing YAP1-S127A, a nonphosphorylable mutant form of YAP1. Numbers of invading cells, which passed through a Transwell over 6 h of incubation, are shown. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( J ) Results are from autophagy-proficient (sh Ctrl ) or autophagy-deficient (sh ATG5 ) MDA231 cells, transfected with an empty vector or a vector expressing YAP1-S127A, after 3 d of 3D culture. Representative bright field images from ATG5-depleted cells expressing or not YAP1-S127A, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 5 independent experiments). P -values are based on the Student t test.

    Article Snippet: The following antibodies were used for western blotting: anti-AP2A1/adaptin (1:1,000; BD Biosciences, 610502), anti-ACTB/β actin (1:20,000; Sigma-Aldrich, A5441), anti-LC3B (1:1,000; Cell Signaling Technology, 2775), anti-ATG7 (1:1,000; Cell Signaling Technology, 8558), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738), anti-GAPDH (1:1,000; Millipore, MAB374), anti-ATG5 (1:1000; Cosmo Bio, TMD-PH-AT5), anti-YAP1 (1:1,000; Cell Signaling Technology, 4912) and anti-phospho-YAP1 (1:1,000; Cell Signaling Technology, 4911).

    Techniques: Polyacrylamide Gel Electrophoresis, Western Blot, Stable Transfection, Quantitative RT-PCR, Expressing, Cell Culture, Transfection, Invasion Assay, Incubation, Plasmid Preparation, Mutagenesis

    Constitutive recruitment of STAT1 at a subset of STAT1-target promoters in hCAF1 knockdown cells. ChIP assays of untreated hCAF1 kd and control cells were performed using antibodies anti-STAT1 ( A ) and anti-acetyl H4 ( B ). Enriched DNA fragments were quantified by qPCR using specific primers for the indicated promoters with respect to the input DNA and normalized to a reference locus (3′ downstream region of the GAPDH gene). Rabbit IgGs were used as a negative control. ( C–E ) hCAF1 affects chromatin accessibility. ( C ) Schematic representation of GAS, PST1 site and primer positions on IFI27 promoter. ( D ) hCAF1 kd and control cells and ( E ) hCAF1 kd transfected with empty pCIflag (mock) or with pCIflag-mCAF1 (hCAF1 kd -mCAF1, rescued cells expressing mCAF1) were exposed or not to IFNγ for 6 h. Isolated nuclei were then treated with a limiting concentration of PST1 restriction enzyme, which cut GAS containing region in IFI27 promoter. DNA was then purified and the level of intact DNA was determined by qPCR using oligos flanking the GAS element or control region illustrated in ( C ). The experiments were performed in triplicate, expressed as mean values and are representative of at least three independent experiments. Standard deviations are shown.

    Journal: The EMBO Journal

    Article Title: hCAF1/CNOT7 regulates interferon signalling by targeting STAT1

    doi: 10.1038/emboj.2013.11

    Figure Lengend Snippet: Constitutive recruitment of STAT1 at a subset of STAT1-target promoters in hCAF1 knockdown cells. ChIP assays of untreated hCAF1 kd and control cells were performed using antibodies anti-STAT1 ( A ) and anti-acetyl H4 ( B ). Enriched DNA fragments were quantified by qPCR using specific primers for the indicated promoters with respect to the input DNA and normalized to a reference locus (3′ downstream region of the GAPDH gene). Rabbit IgGs were used as a negative control. ( C–E ) hCAF1 affects chromatin accessibility. ( C ) Schematic representation of GAS, PST1 site and primer positions on IFI27 promoter. ( D ) hCAF1 kd and control cells and ( E ) hCAF1 kd transfected with empty pCIflag (mock) or with pCIflag-mCAF1 (hCAF1 kd -mCAF1, rescued cells expressing mCAF1) were exposed or not to IFNγ for 6 h. Isolated nuclei were then treated with a limiting concentration of PST1 restriction enzyme, which cut GAS containing region in IFI27 promoter. DNA was then purified and the level of intact DNA was determined by qPCR using oligos flanking the GAS element or control region illustrated in ( C ). The experiments were performed in triplicate, expressed as mean values and are representative of at least three independent experiments. Standard deviations are shown.

    Article Snippet: Anti-phopho Tyr701 STAT1 (#9171S) rabbit polyclonal antibody was purchased from Cell Signaling Technology, the anti-GAPDH (clone 6C5) was from Biodesign International and the anti-acetyl-Histone H4 (06-598) from Millipore.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Transfection, Expressing, Isolation, Concentration Assay, Purification

    Senescence of P8 cells requires p53. (A) Left: Representative immunoblots for p16, p19, and p53 in P2 neurospheres from 2-m R1 and P8 mice. (B) Densitometric quantification of p16, p19, and p53 relative to Gapdh levels (R1 n = 7, P8 n = 7). (C) Percentage of cells with γ-H2AX + foci. Positive control (C+) is a doxorubicin-treated (0.5 μg/ml, 6 h) neurosphere culture. (D) Left : Representative immunoblot for phospho-p53 in P2 neurospheres from 2-m R1 and P8 mice. Right : Densitometric quantification of pp53 relative to Gapdh levels (R1 n = 3, P8 n = 3). (E) Treatment with 20 μ m p53 inhibitor PFTα or 10 μ m ATM inhibitor KU55933 prevents the P8 senescent phenotype. (F) SA β-gal labeling of P8 cells infected with a control or with a p53 shRNA-carrying retrovirus (R1 n = 4, P8 n = 4). (G) Representative images of p53 shRNA and control-infected cultures. Upper panels : phase contrast. Lower panels : SA β-gal staining. Data are shown as mean ± SEM of the indicated number of cultures ( n ) from each strain (* P

    Journal: Aging Cell

    Article Title: Regulation of the p19Arf/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice

    doi: 10.1111/acel.12328

    Figure Lengend Snippet: Senescence of P8 cells requires p53. (A) Left: Representative immunoblots for p16, p19, and p53 in P2 neurospheres from 2-m R1 and P8 mice. (B) Densitometric quantification of p16, p19, and p53 relative to Gapdh levels (R1 n = 7, P8 n = 7). (C) Percentage of cells with γ-H2AX + foci. Positive control (C+) is a doxorubicin-treated (0.5 μg/ml, 6 h) neurosphere culture. (D) Left : Representative immunoblot for phospho-p53 in P2 neurospheres from 2-m R1 and P8 mice. Right : Densitometric quantification of pp53 relative to Gapdh levels (R1 n = 3, P8 n = 3). (E) Treatment with 20 μ m p53 inhibitor PFTα or 10 μ m ATM inhibitor KU55933 prevents the P8 senescent phenotype. (F) SA β-gal labeling of P8 cells infected with a control or with a p53 shRNA-carrying retrovirus (R1 n = 4, P8 n = 4). (G) Representative images of p53 shRNA and control-infected cultures. Upper panels : phase contrast. Lower panels : SA β-gal staining. Data are shown as mean ± SEM of the indicated number of cultures ( n ) from each strain (* P

    Article Snippet: The membranes were blocked 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline supplied with 0.1% Tween 20 (TBS-T) and incubated with rabbit anti-p53 (1:500, Novocastra), ac-p53 (1:1000, Cell Signaling), ac-H3 (1:10 000, Sigma), p16 (1:200, Santa Cruz) or pp53 (S18) (1:500, RyD), rat anti-p19 (1:1000, Abcam), or mouse anti-Gapdh (1:1500, Millipore) antibodies in blocking buffer, o/n at 4 °C.

    Techniques: Western Blot, Mouse Assay, Positive Control, Labeling, Infection, shRNA, Staining

    Inhibition of HDACs in neurospheres induces senescence. (A) Left: Representative immunoblot for Ac-p53 and p19 in R1 neurospheres treated with TSA or VPA. Right : Densitometric quantification reveals a TSA and VPA dose-dependent increase in Ac-p53 and p19 protein levels, relative to Gapdh (in arbitrary units, a.u.). (B) Percentage of BrdU-positive cells in P8, R1, and R1 TSA- or VPA-treated neurospheres. (C) Secondary spheres from R1 mice treated with vehicle (DMSO), 50 n m TSA, or 4 m m VPA. Insets : TSA- and VPA-treated R1 neurospheres exhibit SA β-gal staining. (D) SA β-gal labeling of P8 cells infected with control or p53 shRNA-carrying retroviruses and treated with DMSO or 50 n m TSA (R1 n = 5, P8 n = 5). (E) Fold changes in the number of neurospheres, of SA β-gal + labeling, in the number of S100β + cells, and the level of S100β mRNA in TSA-treated relative to untreated cultures in C57BL/6 wild-type and p53 or p19 mutant mice. (F) Senescent phenotype of P8 cultures is rescued by treatment with 50 μ m anacardic acid (AA). Data are shown as mean ± SEM of 3 independent cultures from each strain or treatment (* P

    Journal: Aging Cell

    Article Title: Regulation of the p19Arf/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice

    doi: 10.1111/acel.12328

    Figure Lengend Snippet: Inhibition of HDACs in neurospheres induces senescence. (A) Left: Representative immunoblot for Ac-p53 and p19 in R1 neurospheres treated with TSA or VPA. Right : Densitometric quantification reveals a TSA and VPA dose-dependent increase in Ac-p53 and p19 protein levels, relative to Gapdh (in arbitrary units, a.u.). (B) Percentage of BrdU-positive cells in P8, R1, and R1 TSA- or VPA-treated neurospheres. (C) Secondary spheres from R1 mice treated with vehicle (DMSO), 50 n m TSA, or 4 m m VPA. Insets : TSA- and VPA-treated R1 neurospheres exhibit SA β-gal staining. (D) SA β-gal labeling of P8 cells infected with control or p53 shRNA-carrying retroviruses and treated with DMSO or 50 n m TSA (R1 n = 5, P8 n = 5). (E) Fold changes in the number of neurospheres, of SA β-gal + labeling, in the number of S100β + cells, and the level of S100β mRNA in TSA-treated relative to untreated cultures in C57BL/6 wild-type and p53 or p19 mutant mice. (F) Senescent phenotype of P8 cultures is rescued by treatment with 50 μ m anacardic acid (AA). Data are shown as mean ± SEM of 3 independent cultures from each strain or treatment (* P

    Article Snippet: The membranes were blocked 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline supplied with 0.1% Tween 20 (TBS-T) and incubated with rabbit anti-p53 (1:500, Novocastra), ac-p53 (1:1000, Cell Signaling), ac-H3 (1:10 000, Sigma), p16 (1:200, Santa Cruz) or pp53 (S18) (1:500, RyD), rat anti-p19 (1:1000, Abcam), or mouse anti-Gapdh (1:1500, Millipore) antibodies in blocking buffer, o/n at 4 °C.

    Techniques: Inhibition, Mouse Assay, Staining, Labeling, Infection, shRNA, Mutagenesis