gapdh  (Millipore)


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    Name:
    Control
    Description:
    Particle size and shape Vehicle control mean particle size is 80 to 90 nm with very small poly dispersity valueFormulation The long circulating pegylated liposomal carrier is composed of N carbonyl ethoxypolyethylene glycol 2000 1 2 distearoyl sn glycero 3 phosphoethanolamine sodium salt MPEG DSPE 3 19 mg ml fully hydrogenated soy phosphatidylcholine HSPC 9 58 mg ml cholesterol 3 19 mg ml ammonium sulfate approximately 2 mg ml histidine as a buffer hydrochloric acid or sodium hydroxide for pH control and sucrose to maintain isotonicity The Control is provided as a sterile translucent liposomal dispersion in single use 2 ml or 5 ml vials Buffer 10 mM histidine buffer pH 6 5 6 8 with 10 w v sucrose
    Catalog Number:
    300103s
    Price:
    None
    Applications:
    Intravenous (i.v.) injection in flank or orthotopic animal models of cancer.
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    Structured Review

    Millipore gapdh
    Detection of CRMP-5 and PKB in retinal proteome through <t>microarray.</t> CRMP-5 was detected significantly higher in the experimental group compared to the control group. Additionally, PKB was detected insignificantly higher in the experimental group. <t>GAPDH</t> served as loading control (n = 6, p
    Particle size and shape Vehicle control mean particle size is 80 to 90 nm with very small poly dispersity valueFormulation The long circulating pegylated liposomal carrier is composed of N carbonyl ethoxypolyethylene glycol 2000 1 2 distearoyl sn glycero 3 phosphoethanolamine sodium salt MPEG DSPE 3 19 mg ml fully hydrogenated soy phosphatidylcholine HSPC 9 58 mg ml cholesterol 3 19 mg ml ammonium sulfate approximately 2 mg ml histidine as a buffer hydrochloric acid or sodium hydroxide for pH control and sucrose to maintain isotonicity The Control is provided as a sterile translucent liposomal dispersion in single use 2 ml or 5 ml vials Buffer 10 mM histidine buffer pH 6 5 6 8 with 10 w v sucrose
    https://www.bioz.com/result/gapdh/product/Millipore
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    gapdh - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Neuroprotective and neuroregenerative effects of CRMP-5 on retinal ganglion cells in an experimental in vivo and in vitro model of glaucoma"

    Article Title: Neuroprotective and neuroregenerative effects of CRMP-5 on retinal ganglion cells in an experimental in vivo and in vitro model of glaucoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207190

    Detection of CRMP-5 and PKB in retinal proteome through microarray. CRMP-5 was detected significantly higher in the experimental group compared to the control group. Additionally, PKB was detected insignificantly higher in the experimental group. GAPDH served as loading control (n = 6, p
    Figure Legend Snippet: Detection of CRMP-5 and PKB in retinal proteome through microarray. CRMP-5 was detected significantly higher in the experimental group compared to the control group. Additionally, PKB was detected insignificantly higher in the experimental group. GAPDH served as loading control (n = 6, p

    Techniques Used: Microarray

    2) Product Images from "Nuclear relocalisation of cytoplasmic poly(A)-binding proteins PABP1 and PABP4 in response to UV irradiation reveals mRNA-dependent export of metazoan PABPs"

    Article Title: Nuclear relocalisation of cytoplasmic poly(A)-binding proteins PABP1 and PABP4 in response to UV irradiation reveals mRNA-dependent export of metazoan PABPs

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.087692

    PABP1 and PABP4 are not determinants of poly(A) RNA localisation. ( A ) Knockdown of PABP1 and PABP4 protein in siRNA-treated HeLa cells was confirmed by western blotting. GAPDH was used as a loading control. ( B ) After transfection with a control siRNA (yellow bars) or PABP1 and PABP4-specific siRNAs (orange bars), HeLa cells were treated with 50 J/m 2 UV followed by 15 hours incubation, and adherent cells assayed for annexin V–FITC and PI staining by flow cytometry. Percentage annexin V-negative and PI-negative cells are shown. Data represented as means ± s.e.m. of three independent experiments. ( C ) Control or PABP1 and PABP4 siRNA-transfected HeLa cells were treated with 50 J/m 2 UV followed by 15 hours incubation or with 0.5 mM sodium arsenite for 1 hour. Poly(A) RNA was detected by FISH using a Cy3–oligo(dT) 40 probe (red). DNA was stained by DAPI (blue). Scale bar: 20 μm. ( D ) siRNA-transfected HeLa cells were treated with 50 J/m 2 UV followed by 3 hours incubation or with 0.5 mM sodium arsenite for 1 hour. The cells were then fixed and G3BP (red) detected by immunofluorescence. DNA was stained with DAPI (blue). Scale bar: 20 μm.
    Figure Legend Snippet: PABP1 and PABP4 are not determinants of poly(A) RNA localisation. ( A ) Knockdown of PABP1 and PABP4 protein in siRNA-treated HeLa cells was confirmed by western blotting. GAPDH was used as a loading control. ( B ) After transfection with a control siRNA (yellow bars) or PABP1 and PABP4-specific siRNAs (orange bars), HeLa cells were treated with 50 J/m 2 UV followed by 15 hours incubation, and adherent cells assayed for annexin V–FITC and PI staining by flow cytometry. Percentage annexin V-negative and PI-negative cells are shown. Data represented as means ± s.e.m. of three independent experiments. ( C ) Control or PABP1 and PABP4 siRNA-transfected HeLa cells were treated with 50 J/m 2 UV followed by 15 hours incubation or with 0.5 mM sodium arsenite for 1 hour. Poly(A) RNA was detected by FISH using a Cy3–oligo(dT) 40 probe (red). DNA was stained by DAPI (blue). Scale bar: 20 μm. ( D ) siRNA-transfected HeLa cells were treated with 50 J/m 2 UV followed by 3 hours incubation or with 0.5 mM sodium arsenite for 1 hour. The cells were then fixed and G3BP (red) detected by immunofluorescence. DNA was stained with DAPI (blue). Scale bar: 20 μm.

    Techniques Used: Western Blot, Transfection, Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence In Situ Hybridization, Immunofluorescence

    3) Product Images from "Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma"

    Article Title: Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103835

    Investigation of mechanisms underlying NMR-detected changes in the levels of choline metabolites and lactate following treatment of SF188 pediatric glioblastoma cells with PI-103 (5×GI 50 ). (A) Representative Western blots showing changes in protein expression levels of enzymes involved in choline metabolism (CHKA) and glucose metabolism including: GLUT1, HK2 and LDHA, at selected time points post treatment with PI-103. GAPDH was used as a loading control. (B) Quantitative measurement of 1 H-NMR detected percentage changes in the levels of lactate (Lac, internal external) and glucose (external) at selected time points post treatment with PI-103 relative to controls, 8 hours n = 2. Results are expressed as percentage of treated to control and presented as the mean ± SD (error bars). Statistically significant different from the control *p≤0.05, **p
    Figure Legend Snippet: Investigation of mechanisms underlying NMR-detected changes in the levels of choline metabolites and lactate following treatment of SF188 pediatric glioblastoma cells with PI-103 (5×GI 50 ). (A) Representative Western blots showing changes in protein expression levels of enzymes involved in choline metabolism (CHKA) and glucose metabolism including: GLUT1, HK2 and LDHA, at selected time points post treatment with PI-103. GAPDH was used as a loading control. (B) Quantitative measurement of 1 H-NMR detected percentage changes in the levels of lactate (Lac, internal external) and glucose (external) at selected time points post treatment with PI-103 relative to controls, 8 hours n = 2. Results are expressed as percentage of treated to control and presented as the mean ± SD (error bars). Statistically significant different from the control *p≤0.05, **p

    Techniques Used: Nuclear Magnetic Resonance, Western Blot, Expressing

    4) Product Images from "Expression of androgen receptor splice variants in clinical breast cancers"

    Article Title: Expression of androgen receptor splice variants in clinical breast cancers

    Journal: Oncotarget

    doi:

    AR-V7 regulates the growth of MDA-MB-453 cells and response to enzalutamide A–B. MDA-MB-453 cells were transfected with siRNAs specific for AR-FL and AR-V7 or a control siRNA and growth was assessed in androgen-replete media using MTT (A) and Trypan Blue (B) growth assays. Values are the mean (±SEM) of 3 biological replicates; results are representative of three independent experiments. C. MDA-MB-453 cells were transduced with lentivirus designed to overexpress AR-V7 or GFP (negative control) and cell growth assessed using Crystal Violet assays in response to the enzalutamide. The relative change in growth compared to vehicle control is shown at 6 and 9 days. Values are the mean (±SEM) of 6 biological replicates comprising two independent experiments. D. Treatment with AR antagonists induces AR gene transcription in MDA-MB-453 cells. Cells were grown in androgen depleted media and treated with 10 nM DHT, 10 μM bicalutamide, 10 μM enzalutamide or vehicle control for 24 h. AR-FL and AR-V7 mRNA was measured by qRT-PCR. Values are normalized to GAPDH and represent the mean (± SEM) of triplicate samples, with vehicle control treatment set to 1. Results are representative of three independent experiments. E. Matched samples from (D) were analyzed by Western blotting using AR-V7 (Precision Biosciences), AR (N20) and GAPDH (loading control) antibodies. F. Human breast tumor explants were cultured with vehicle (DMSO) or enzalutamide (50 μM) for 48 h. AR-FL and AR-V7 transcripts were measured by qRT-PCR and normalized to GAPDH . Values are expressed as fold-change relative to DMSO for each individual tumor. Statistically significant differences compared to the control treatment were assessed using a Wilcoxon signed rank test; p values are shown. Four of the tumors did not express AR-V7 (shown by a # symbol).
    Figure Legend Snippet: AR-V7 regulates the growth of MDA-MB-453 cells and response to enzalutamide A–B. MDA-MB-453 cells were transfected with siRNAs specific for AR-FL and AR-V7 or a control siRNA and growth was assessed in androgen-replete media using MTT (A) and Trypan Blue (B) growth assays. Values are the mean (±SEM) of 3 biological replicates; results are representative of three independent experiments. C. MDA-MB-453 cells were transduced with lentivirus designed to overexpress AR-V7 or GFP (negative control) and cell growth assessed using Crystal Violet assays in response to the enzalutamide. The relative change in growth compared to vehicle control is shown at 6 and 9 days. Values are the mean (±SEM) of 6 biological replicates comprising two independent experiments. D. Treatment with AR antagonists induces AR gene transcription in MDA-MB-453 cells. Cells were grown in androgen depleted media and treated with 10 nM DHT, 10 μM bicalutamide, 10 μM enzalutamide or vehicle control for 24 h. AR-FL and AR-V7 mRNA was measured by qRT-PCR. Values are normalized to GAPDH and represent the mean (± SEM) of triplicate samples, with vehicle control treatment set to 1. Results are representative of three independent experiments. E. Matched samples from (D) were analyzed by Western blotting using AR-V7 (Precision Biosciences), AR (N20) and GAPDH (loading control) antibodies. F. Human breast tumor explants were cultured with vehicle (DMSO) or enzalutamide (50 μM) for 48 h. AR-FL and AR-V7 transcripts were measured by qRT-PCR and normalized to GAPDH . Values are expressed as fold-change relative to DMSO for each individual tumor. Statistically significant differences compared to the control treatment were assessed using a Wilcoxon signed rank test; p values are shown. Four of the tumors did not express AR-V7 (shown by a # symbol).

    Techniques Used: Multiple Displacement Amplification, Transfection, MTT Assay, Transduction, Negative Control, Quantitative RT-PCR, Western Blot, Cell Culture

    Expression of AR-V7 in breast cancer cell line models A–B. qRT-PCR was used to quantify AR-V7 (A) and AR-FL (B) in a panel of 8 breast cancer cell lines and 2 prostate cancer cell line controls (22Rv1 and VCaP). AR-V7 levels as a percentage of AR-FL transcript are shown above the columns in panel A. Values are the mean (± SEM) of triplicate samples. C. Protein lysates were collected from the indicated cell lines used above and subjected to Western blot analysis using AR-V7 (Precision Biosciences), AR (N20) and GAPDH (loading control) antibodies. Short and long exposures are shown. Note that AR-FL from 22Rv1 cells migrates more slowly because it contains two copies of the second zinc finger domain as a result of a genomic duplication. D. MDA-MB-453 and 22Rv1 cells were transfected with a non-specific control siRNA (NC) or siRNAs specific for AR-FL or AR-V7. After 72 h, protein lysates were analyzed by Western blotting as in (C).
    Figure Legend Snippet: Expression of AR-V7 in breast cancer cell line models A–B. qRT-PCR was used to quantify AR-V7 (A) and AR-FL (B) in a panel of 8 breast cancer cell lines and 2 prostate cancer cell line controls (22Rv1 and VCaP). AR-V7 levels as a percentage of AR-FL transcript are shown above the columns in panel A. Values are the mean (± SEM) of triplicate samples. C. Protein lysates were collected from the indicated cell lines used above and subjected to Western blot analysis using AR-V7 (Precision Biosciences), AR (N20) and GAPDH (loading control) antibodies. Short and long exposures are shown. Note that AR-FL from 22Rv1 cells migrates more slowly because it contains two copies of the second zinc finger domain as a result of a genomic duplication. D. MDA-MB-453 and 22Rv1 cells were transfected with a non-specific control siRNA (NC) or siRNAs specific for AR-FL or AR-V7. After 72 h, protein lysates were analyzed by Western blotting as in (C).

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Multiple Displacement Amplification, Transfection

    5) Product Images from "Sudden Infant Death Syndrome in Mice with an Inherited Mutation in RyR2"

    Article Title: Sudden Infant Death Syndrome in Mice with an Inherited Mutation in RyR2

    Journal: Circulation. Arrhythmia and electrophysiology

    doi: 10.1161/CIRCEP.109.894683

    Expression levels of calcium handling proteins are unaltered in neonatal R176Q/+ mice A , Representative examples of RT-PCR products for the genes encoding Cav1.2, RyR2, SERCA2a, PLB, and NCX1, and the loading control (60S ribosomal protein L7). Summary bar graphs show that mRNA expression levels of these Ca 2+ -handling proteins are unaltered in neonatal R176Q/+ knock-in mice after normalization to the L7 housekeeping gene (n=4, each genotype). B , Representative examples of Western blots for proteins Cav1.2, RyR2, SERCA2a, PLB, NCX1, and the loading control GAPDH. Quantification of protein levels normalized to GAPDH expression reveals no significant differences in the levels of these major Ca 2+ -handling proteins in 1-day old WT and R176Q/+ knock-in mice.
    Figure Legend Snippet: Expression levels of calcium handling proteins are unaltered in neonatal R176Q/+ mice A , Representative examples of RT-PCR products for the genes encoding Cav1.2, RyR2, SERCA2a, PLB, and NCX1, and the loading control (60S ribosomal protein L7). Summary bar graphs show that mRNA expression levels of these Ca 2+ -handling proteins are unaltered in neonatal R176Q/+ knock-in mice after normalization to the L7 housekeeping gene (n=4, each genotype). B , Representative examples of Western blots for proteins Cav1.2, RyR2, SERCA2a, PLB, NCX1, and the loading control GAPDH. Quantification of protein levels normalized to GAPDH expression reveals no significant differences in the levels of these major Ca 2+ -handling proteins in 1-day old WT and R176Q/+ knock-in mice.

    Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Knock-In, Western Blot

    6) Product Images from "Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB"

    Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00924

    Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p
    Figure Legend Snippet: Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p

    Techniques Used: Western Blot, Injection

    7) Product Images from "Intracellular fatty acids suppress ?-adrenergic induction of PKA-targeted gene expression in white adipocytes"

    Article Title: Intracellular fatty acids suppress ?-adrenergic induction of PKA-targeted gene expression in white adipocytes

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    doi: 10.1152/ajpendo.00039.2011

    Knockdown of adipose triglyceride lipase (ATGL) potentiates β-AR induction of PKA-targeted genes. A : Western blot for ATGL, HSL, and GAPDH from 3 separate wells per small interfering (si)RNA. GAPDH serves as a loading control. B : glycerol levels
    Figure Legend Snippet: Knockdown of adipose triglyceride lipase (ATGL) potentiates β-AR induction of PKA-targeted genes. A : Western blot for ATGL, HSL, and GAPDH from 3 separate wells per small interfering (si)RNA. GAPDH serves as a loading control. B : glycerol levels

    Techniques Used: Western Blot

    8) Product Images from "Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine"

    Article Title: Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156877

    Proliferation and apoptosis analysis of the small intestine. A: Quantification of the total number of cells per crypt. A minimum of 50 crypts were quantitated per animal of each genotype and mean values are shown + SEM. Genotypes were: GSK3α +/+ ;GSK3β +/+ (n = 3), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 3). No statistically significant difference was observed using the Student’s t-test. B: Immunohistochemical staining of WT/WT and KI/KI small intestinal crypts using antibodies for BrdU or phospho-Histone H3. Scale bars, 20μm. C: Quantification of BrdU-positive cells per crypt within the WT/WT and KI/KI small intestine. Staining in 50 crypts of three mice of each genotype was quantified. No statistically significant difference was observed using the Student’s t-test. D: The position of BrdU-positive cells within the WT/WT and KI/KI crypts was quantified. Position 0 represents the base of the crypt. A minimum of 50 crypts were quantified in three mice of each genotype. No statistically significant difference was observed using the Student’s t-test. E: Immunohistochemical staining of WT/WT and KI/KI villi using an antibody for cleaved caspase 3. Scale bars, 50μm. F: Western blot analysis of small intestinal protein samples from WT/WT or KI/KI mice using antibodies for phosphorylated GSK3α/β, cleaved PARP or cleaved caspase-3. Immunoblotting for ERK2 and GAPDH was used to determine equal protein loading.
    Figure Legend Snippet: Proliferation and apoptosis analysis of the small intestine. A: Quantification of the total number of cells per crypt. A minimum of 50 crypts were quantitated per animal of each genotype and mean values are shown + SEM. Genotypes were: GSK3α +/+ ;GSK3β +/+ (n = 3), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 3). No statistically significant difference was observed using the Student’s t-test. B: Immunohistochemical staining of WT/WT and KI/KI small intestinal crypts using antibodies for BrdU or phospho-Histone H3. Scale bars, 20μm. C: Quantification of BrdU-positive cells per crypt within the WT/WT and KI/KI small intestine. Staining in 50 crypts of three mice of each genotype was quantified. No statistically significant difference was observed using the Student’s t-test. D: The position of BrdU-positive cells within the WT/WT and KI/KI crypts was quantified. Position 0 represents the base of the crypt. A minimum of 50 crypts were quantified in three mice of each genotype. No statistically significant difference was observed using the Student’s t-test. E: Immunohistochemical staining of WT/WT and KI/KI villi using an antibody for cleaved caspase 3. Scale bars, 50μm. F: Western blot analysis of small intestinal protein samples from WT/WT or KI/KI mice using antibodies for phosphorylated GSK3α/β, cleaved PARP or cleaved caspase-3. Immunoblotting for ERK2 and GAPDH was used to determine equal protein loading.

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

    9) Product Images from "Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: a novel therapeutic lead to treat frontotemporal dementia"

    Article Title: Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: a novel therapeutic lead to treat frontotemporal dementia

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-016-0114-3

    The transcription factor EB (TFEB) does not mediate trehalose-induced upregulation of PGRN. a GRN mRNA levels were quantified from wild-type HeLa (WT) cells or HeLa cells stably over-expressing TFEB-GFP (TFEB-GFP) ( n = 3 independent experiments run in duplicate). b WT or TFEB-GFP HeLa cells were fractionated into cytoplasmic/membrane and nuclear extracts and immunoblotted for total TFEB and PGRN. GAPDH and H3 were used to verify fractionation efficiency and were used as loading controls. Asterisk (*) denotes non-specific band in nuclear fraction. c Quantification of PGRN expression in WT or TFEB-GFP HeLa lysates in b. ( n = 3 independent experiments). d TFEB-GFP HeLa cells were treated with trehalose (100 mM) or Torin1 (250 nM) for 0, 2, or 24 h and imaged live with a fluorescent microscope to visualize TFEB-GFP localization. e TFEB-GFP HeLa cells were treated with vehicle, trehalose (100 mM), or Torin1 (250 nM) for 24 h and then fractionated into cytoplasmic/membrane and nuclear extracts and immunoblotted for total TFEB and PGRN. Asterisk (*) denotes non-specific band in nuclear fraction. f Quantification of nuclear TFEB (top) and cytoplasmic/membrane PGRN (bottom) from the immunoblots in e. ( n = 3 independent experiments). g Immunoblots from whole-cell lysates of HAP1 WT and TFEB KO cells showing absence of TFEB expression. h GRN mRNA levels were quantified from HAP1 wild-type (WT) and TFEB knock-out (TFEB KO) cell lines after treatment with vehicle or trehalose (100 mM) for 18 h ( n = 3 independent experiments run in duplicate). i HAP1 WT and TFEB KO cells were treated with vehicle or trehalose (100 mM) for 24 h and then fractionated into cytoplasmic/membrane and nuclear extracts. Immunoblots for PGRN (cytoplasmic/membrane) and TFEB (nuclear) are shown. j Quantification of PGRN (top) and TFEB (bottom) from the immunoblots in i ( n = 3 independent experiments). In all graphs, the bars represent the mean ± SEM. For a, c; *differs from WT, P
    Figure Legend Snippet: The transcription factor EB (TFEB) does not mediate trehalose-induced upregulation of PGRN. a GRN mRNA levels were quantified from wild-type HeLa (WT) cells or HeLa cells stably over-expressing TFEB-GFP (TFEB-GFP) ( n = 3 independent experiments run in duplicate). b WT or TFEB-GFP HeLa cells were fractionated into cytoplasmic/membrane and nuclear extracts and immunoblotted for total TFEB and PGRN. GAPDH and H3 were used to verify fractionation efficiency and were used as loading controls. Asterisk (*) denotes non-specific band in nuclear fraction. c Quantification of PGRN expression in WT or TFEB-GFP HeLa lysates in b. ( n = 3 independent experiments). d TFEB-GFP HeLa cells were treated with trehalose (100 mM) or Torin1 (250 nM) for 0, 2, or 24 h and imaged live with a fluorescent microscope to visualize TFEB-GFP localization. e TFEB-GFP HeLa cells were treated with vehicle, trehalose (100 mM), or Torin1 (250 nM) for 24 h and then fractionated into cytoplasmic/membrane and nuclear extracts and immunoblotted for total TFEB and PGRN. Asterisk (*) denotes non-specific band in nuclear fraction. f Quantification of nuclear TFEB (top) and cytoplasmic/membrane PGRN (bottom) from the immunoblots in e. ( n = 3 independent experiments). g Immunoblots from whole-cell lysates of HAP1 WT and TFEB KO cells showing absence of TFEB expression. h GRN mRNA levels were quantified from HAP1 wild-type (WT) and TFEB knock-out (TFEB KO) cell lines after treatment with vehicle or trehalose (100 mM) for 18 h ( n = 3 independent experiments run in duplicate). i HAP1 WT and TFEB KO cells were treated with vehicle or trehalose (100 mM) for 24 h and then fractionated into cytoplasmic/membrane and nuclear extracts. Immunoblots for PGRN (cytoplasmic/membrane) and TFEB (nuclear) are shown. j Quantification of PGRN (top) and TFEB (bottom) from the immunoblots in i ( n = 3 independent experiments). In all graphs, the bars represent the mean ± SEM. For a, c; *differs from WT, P

    Techniques Used: Stable Transfection, Expressing, Fractionation, Microscopy, Western Blot, Knock-Out

    10) Product Images from "Nile Tilapia Derived Antimicrobial Peptide TP4 Exerts Antineoplastic Activity Through Microtubule Disruption"

    Article Title: Nile Tilapia Derived Antimicrobial Peptide TP4 Exerts Antineoplastic Activity Through Microtubule Disruption

    Journal: Marine Drugs

    doi: 10.3390/md16120462

    TP4 disrupts the microtubule network in cancer cells. ( A ) Cells were stained for α-Tubulin and F-actin. Hoechst33342 was used to stain nuclei. Arrows indicate cells with negative α-Tubulin staining. Bar: 20 μm. ( B ) Total lysates from cells with mock (labeled C) or TP4 treatment (labeled T) were analyzed by Western blot using antibodies against GAPDH and α-Tubulin. Quantification of the α-Tubulin signal normalized to GAPDH is shown. Mr is the molecular weight. ( C ) A549 cells pretreated with 20 μM nocodazole were stained for α-Tubulin (white) at 0, 30, and 60 minutes after nocodazole washout. Hoechst33342 was used to stain nuclei (blue). Arrows indicate cells with defective microtubule regrowth. Bar: 20 μm ( D ) Quantification of the number of cells with Tubulin-positive and Tubulin-negative staining. Results are presented as mean ± SD. N = 3, two-way analysis of variance (ANOVA): ** p
    Figure Legend Snippet: TP4 disrupts the microtubule network in cancer cells. ( A ) Cells were stained for α-Tubulin and F-actin. Hoechst33342 was used to stain nuclei. Arrows indicate cells with negative α-Tubulin staining. Bar: 20 μm. ( B ) Total lysates from cells with mock (labeled C) or TP4 treatment (labeled T) were analyzed by Western blot using antibodies against GAPDH and α-Tubulin. Quantification of the α-Tubulin signal normalized to GAPDH is shown. Mr is the molecular weight. ( C ) A549 cells pretreated with 20 μM nocodazole were stained for α-Tubulin (white) at 0, 30, and 60 minutes after nocodazole washout. Hoechst33342 was used to stain nuclei (blue). Arrows indicate cells with defective microtubule regrowth. Bar: 20 μm ( D ) Quantification of the number of cells with Tubulin-positive and Tubulin-negative staining. Results are presented as mean ± SD. N = 3, two-way analysis of variance (ANOVA): ** p

    Techniques Used: Staining, Labeling, Western Blot, Molecular Weight, Negative Staining

    11) Product Images from "The prognostic role and reduced expression of FOXJ2 in human hepatocellular carcinoma"

    Article Title: The prognostic role and reduced expression of FOXJ2 in human hepatocellular carcinoma

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5261

    FOXJ2 was downregulated in human HCC tissues and HCC cell lines. (A) Western blot analysis was performed to detect the expression of FOXJ2 in 8 representative paired HCC tumor tissues (T) and adjacent non-tumor tissues (N). GAPDH was used as a loading control. (B) The band intensity of FOXJ2 and GAPDH was quantified and the FOXJ2 protein level was normalized to the GAPDH protein. The data are presented as the mean ± standard deviation of three independent experiments ( * P
    Figure Legend Snippet: FOXJ2 was downregulated in human HCC tissues and HCC cell lines. (A) Western blot analysis was performed to detect the expression of FOXJ2 in 8 representative paired HCC tumor tissues (T) and adjacent non-tumor tissues (N). GAPDH was used as a loading control. (B) The band intensity of FOXJ2 and GAPDH was quantified and the FOXJ2 protein level was normalized to the GAPDH protein. The data are presented as the mean ± standard deviation of three independent experiments ( * P

    Techniques Used: Western Blot, Expressing, Standard Deviation

    FOXJ2 knockdown promoted cell proliferation. (A) Western blot analysis indicated that siRNA treatment of FOXJ2 markedly reduced the FOXJ2 levels 48 h subsequent to siRNA transfection in three HCC cells in comparison with cells transfected with negative control and mock treatments. (B) The ratio of FOXJ2 protein to GAPDH expression measured by densitometry. Data are presented as the mean ± standard error. (C) A CCK-8 assay indicated that FOXJ2 knockdown in HepG2 cell promoted cell proliferation. CCK-8 reagents were added to the medium and incubated for additional 2 h. Absorbance was measured at each indicated time (0, 12, 24, 48 and 72 h). Each time point was derived from three independent experiments. The data are presented as the mean ± standard deviation for three experiments. * P
    Figure Legend Snippet: FOXJ2 knockdown promoted cell proliferation. (A) Western blot analysis indicated that siRNA treatment of FOXJ2 markedly reduced the FOXJ2 levels 48 h subsequent to siRNA transfection in three HCC cells in comparison with cells transfected with negative control and mock treatments. (B) The ratio of FOXJ2 protein to GAPDH expression measured by densitometry. Data are presented as the mean ± standard error. (C) A CCK-8 assay indicated that FOXJ2 knockdown in HepG2 cell promoted cell proliferation. CCK-8 reagents were added to the medium and incubated for additional 2 h. Absorbance was measured at each indicated time (0, 12, 24, 48 and 72 h). Each time point was derived from three independent experiments. The data are presented as the mean ± standard deviation for three experiments. * P

    Techniques Used: Western Blot, Transfection, Negative Control, Expressing, CCK-8 Assay, Incubation, Derivative Assay, Standard Deviation

    12) Product Images from "NFAT regulates the expression of AIF-1 and IRT-1: yin and yang splice variants of neointima formation and atherosclerosis"

    Article Title: NFAT regulates the expression of AIF-1 and IRT-1: yin and yang splice variants of neointima formation and atherosclerosis

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvr309

    Opposite effects of AIF-1 and IRT-1 on VSMC proliferation and migration. ( A ) Western blots confirming overexpression of AIF-1 (upper panel) or IRT-1 (lower panel) in infected rat VSMCs. GAPDH was used to verify equal loading. ( B ) Effect of AdAIF-1 and
    Figure Legend Snippet: Opposite effects of AIF-1 and IRT-1 on VSMC proliferation and migration. ( A ) Western blots confirming overexpression of AIF-1 (upper panel) or IRT-1 (lower panel) in infected rat VSMCs. GAPDH was used to verify equal loading. ( B ) Effect of AdAIF-1 and

    Techniques Used: Migration, Western Blot, Over Expression, Infection

    13) Product Images from "The c-Jun N-terminal Kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition"

    Article Title: The c-Jun N-terminal Kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition

    Journal: Pain

    doi: 10.1016/j.pain.2009.11.017

    Western blot analysis showing time course of CFA-induced expression of pJNK1, pJNK2, JNK1, and JNK2 in the spinal cord of rats. JNK1 and JNK2 are rapidly activated and maintained for > 14 days in both ipsilateral (A) and contralateral (B) lumbar spinal cord. Low panels in A and B show densities of pJNK1 and pJNK2 bands after being normalized to tubulin. Total levels of JNK1 and JNK2 do not change after inflammation (C). Low panels in C shows densities of JNK1 and JNK2 against GAPDH. *, P
    Figure Legend Snippet: Western blot analysis showing time course of CFA-induced expression of pJNK1, pJNK2, JNK1, and JNK2 in the spinal cord of rats. JNK1 and JNK2 are rapidly activated and maintained for > 14 days in both ipsilateral (A) and contralateral (B) lumbar spinal cord. Low panels in A and B show densities of pJNK1 and pJNK2 bands after being normalized to tubulin. Total levels of JNK1 and JNK2 do not change after inflammation (C). Low panels in C shows densities of JNK1 and JNK2 against GAPDH. *, P

    Techniques Used: Western Blot, Expressing

    14) Product Images from "Biliary tract instillation of a SMAC mimetic induces TRAIL-dependent acute sclerosing cholangitis-like injury in mice"

    Article Title: Biliary tract instillation of a SMAC mimetic induces TRAIL-dependent acute sclerosing cholangitis-like injury in mice

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.459

    SM-induced upregulation of pro-inflammatory cytokines is mediated by activation of the non-canonical NF- κ B pathway. ( a ) Immunoblot analysis showing expression of cIAP-1, cIAP-2, NIK, I κ B α , PARP, IL-6 and actin (loading control) in H69 cells treated with TL32711 for the indicated times. ( b ) Immunoblot analysis showing expression of RIP1, NIK, NF- κ B2, actin (loading control), I κ B α and GAPDH (loading control) in H69 and RIP1 −/− H69 cells treated with TL32711 for the indicated times. ( c ) IL6 , IL8 and MCP1/CCL2 gene expression analyzed by qPCR in NHC cell line transiently transfected with siRNA against NF- κ B2 (p100) or scrambled siRNA for 48 h and incubated in the presence or absence (cnt) of TL32711 for 4 h. Efficiency of the knockdown was evaluated by immunoblot (insert). Data are expressed as fold increase over control. Mean±S.E. are depicted from six independent experiments. * P
    Figure Legend Snippet: SM-induced upregulation of pro-inflammatory cytokines is mediated by activation of the non-canonical NF- κ B pathway. ( a ) Immunoblot analysis showing expression of cIAP-1, cIAP-2, NIK, I κ B α , PARP, IL-6 and actin (loading control) in H69 cells treated with TL32711 for the indicated times. ( b ) Immunoblot analysis showing expression of RIP1, NIK, NF- κ B2, actin (loading control), I κ B α and GAPDH (loading control) in H69 and RIP1 −/− H69 cells treated with TL32711 for the indicated times. ( c ) IL6 , IL8 and MCP1/CCL2 gene expression analyzed by qPCR in NHC cell line transiently transfected with siRNA against NF- κ B2 (p100) or scrambled siRNA for 48 h and incubated in the presence or absence (cnt) of TL32711 for 4 h. Efficiency of the knockdown was evaluated by immunoblot (insert). Data are expressed as fold increase over control. Mean±S.E. are depicted from six independent experiments. * P

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Incubation

    15) Product Images from "Effect of Culture Time on the Basal Expression Levels of Drug Transporters in Sandwich-Cultured Primary Rat Hepatocytes S⃞"

    Article Title: Effect of Culture Time on the Basal Expression Levels of Drug Transporters in Sandwich-Cultured Primary Rat Hepatocytes S⃞

    Journal: Drug Metabolism and Disposition

    doi: 10.1124/dmd.111.039545

    Western blotting analysis of the expression of transporter proteins in cultured rat hepatocytes. Western immunoblots were performed using either cultured hepatocyte lysate samples or total liver homogenate (20 mg protein/lane) as indicated under Materials and Methods . GAPDH was used as the loading control, DPP IV as the canalicular marker, and OTF1 as transcriptional control for housekeeping genes.
    Figure Legend Snippet: Western blotting analysis of the expression of transporter proteins in cultured rat hepatocytes. Western immunoblots were performed using either cultured hepatocyte lysate samples or total liver homogenate (20 mg protein/lane) as indicated under Materials and Methods . GAPDH was used as the loading control, DPP IV as the canalicular marker, and OTF1 as transcriptional control for housekeeping genes.

    Techniques Used: Western Blot, Expressing, Cell Culture, Marker

    16) Product Images from "Piperine ameliorates SCA17 neuropathology by reducing ER stress"

    Article Title: Piperine ameliorates SCA17 neuropathology by reducing ER stress

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-018-0236-x

    Piperine reduced ER stress caused by mutant TBP. a , b Stable transfected PC12 cells expressing TBP-13Q ( a ) or TBP-105Q ( b ) were treated with different concentrations (0, 2 or 10 μM) of piperine for 48 h. Different concentrations of tunicamycin (0, 2.5 or 10 μM) were then added for 4 h. Western blotting was performed to examine the levels of XBP1s and CHOP. Untreated cells (ctl) were used as controls. GAPDH was used as a loading control. The ratios of XBP1s or CHOP to GAPDH are presented beneath the blots (n = 3, * P
    Figure Legend Snippet: Piperine reduced ER stress caused by mutant TBP. a , b Stable transfected PC12 cells expressing TBP-13Q ( a ) or TBP-105Q ( b ) were treated with different concentrations (0, 2 or 10 μM) of piperine for 48 h. Different concentrations of tunicamycin (0, 2.5 or 10 μM) were then added for 4 h. Western blotting was performed to examine the levels of XBP1s and CHOP. Untreated cells (ctl) were used as controls. GAPDH was used as a loading control. The ratios of XBP1s or CHOP to GAPDH are presented beneath the blots (n = 3, * P

    Techniques Used: Mutagenesis, Transfection, Expressing, Western Blot, CTL Assay

    17) Product Images from "Secreted hCLCA1 Is a Signaling Molecule That Activates Airway Macrophages"

    Article Title: Secreted hCLCA1 Is a Signaling Molecule That Activates Airway Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083130

    Enhanced macrophage activation with higher concentration of immuno-purified hCLCA1. ( A ) Representative silver stained SDS-PAGE gel showing immuno-purified hCLCA1 and a 2-fold dilution series of lysozyme. ( B ) Densitometry analysis using lysozyme standard curve demonstrated a higher concentration of pure hCLCA1 (9.425 ± 0.335 pg/μL) was immunoprecipitated with an optimized protocol. Result was presented as the mean of 3 samples ± SEM. ( C ) The mRNA expression of cytokines in macrophages stimulated with a higher concentration of hCLCA1 for 48 h was quantified using RT-qPCR. The fold difference was calculated against the corresponding control (immunoprecipitation of eGFP using hCLCA1-N14 antibody). Results were presented as the means of 4 samples ± SEM. ( D ) Representative Western blots showing intracellular IL-1β and GAPDH levels in immuno-purified eGFP or hCLCA1-stimulated macrophages. GAPDH was used as a loading control for densitometry analysis. Immuno-purified hCLCA1-stimulated macrophages had a 2.38 ± 0.21 folds increase in IL-1β protein levels over immuno-purified eGFP-stimulated macrophages (the hCLCA1-induced IL-1β was normalized to the eGFP-induced IL-1β in each sample). Results were presented as the means of 7 samples ± SEM. ( E ) Secreted cytokine protein expression in macrophages stimulated with a higher concentration of immuno-purified hCLCA1 was analyzed using Bio-plex Suspension Array System. The fold difference of each sample was compared against the corresponding control. Results were the means of 3 samples ± SEM. Significant fold differences from corresponding control values are indicated by * ( p
    Figure Legend Snippet: Enhanced macrophage activation with higher concentration of immuno-purified hCLCA1. ( A ) Representative silver stained SDS-PAGE gel showing immuno-purified hCLCA1 and a 2-fold dilution series of lysozyme. ( B ) Densitometry analysis using lysozyme standard curve demonstrated a higher concentration of pure hCLCA1 (9.425 ± 0.335 pg/μL) was immunoprecipitated with an optimized protocol. Result was presented as the mean of 3 samples ± SEM. ( C ) The mRNA expression of cytokines in macrophages stimulated with a higher concentration of hCLCA1 for 48 h was quantified using RT-qPCR. The fold difference was calculated against the corresponding control (immunoprecipitation of eGFP using hCLCA1-N14 antibody). Results were presented as the means of 4 samples ± SEM. ( D ) Representative Western blots showing intracellular IL-1β and GAPDH levels in immuno-purified eGFP or hCLCA1-stimulated macrophages. GAPDH was used as a loading control for densitometry analysis. Immuno-purified hCLCA1-stimulated macrophages had a 2.38 ± 0.21 folds increase in IL-1β protein levels over immuno-purified eGFP-stimulated macrophages (the hCLCA1-induced IL-1β was normalized to the eGFP-induced IL-1β in each sample). Results were presented as the means of 7 samples ± SEM. ( E ) Secreted cytokine protein expression in macrophages stimulated with a higher concentration of immuno-purified hCLCA1 was analyzed using Bio-plex Suspension Array System. The fold difference of each sample was compared against the corresponding control. Results were the means of 3 samples ± SEM. Significant fold differences from corresponding control values are indicated by * ( p

    Techniques Used: Activation Assay, Concentration Assay, Purification, Staining, SDS Page, Immunoprecipitation, Expressing, Quantitative RT-PCR, Western Blot

    18) Product Images from "ELFN2 is a postsynaptic cell adhesion molecule with essential roles in controlling group III mGluRs in the brain and neuropsychiatric behavior"

    Article Title: ELFN2 is a postsynaptic cell adhesion molecule with essential roles in controlling group III mGluRs in the brain and neuropsychiatric behavior

    Journal: Molecular psychiatry

    doi: 10.1038/s41380-019-0512-3

    A. Schematic representation of generation of Elfn2 KO mouse replace exon2 on Elfn2 gene with LacZ-neo cassette for β-galactosidase reporter expression and disruption of ELFN2 protein expression (CDS, coding sequence; UTR, untranslated region). B. Immunohistochemistry for β-galactosidase expression (green) in Elfn2 KO mice coronal section and DAPI nuclear staining (blue). C. Western blot confirming lack of ELFN2 protein expression in Elfn2 KO mouse whole brain homogenate compared to wildtype mouse. GAPDH Western blot provided as loading control. D. Comparative body mass between wildtype and Elfn2 KO mice (p=0.1965, n=8). E. Comparative brain mass between wildtype and Elfn2 KO mice (p=0.5819, n=7). F. Western blotting and densitometric quantification of protein expression for various synaptic machineries compared between wildtype and Elfn2 KO mice: PSD-95 (p=0.1492, n=7), Synaptophysin (p=0.7168, n=7), GluA1 (p=0.6760, n=7), NMDAR 1 (p=0.3416, n=7), NMDAR 2B (p=0.8387, n=7), mGluR5 (p=0.1057, n=7), mGluR2 (p=0.3570, n=5–7), mGluR4 (p=0.0027, n=7), mGluR7 (p=0.0005, n=7), mGluR8 (p=0.0244, n=7), GABA A R α1 (p=0.3671, n=7). G. Quantitative real-time PCR for RNA expression of Elfn2 (p=0.0018, n=4), Grm2 (p > 0.9999, n=4), Grm5 (p > 0.9999, n=4), Grm4 (p=0.6416, n=4), Grm7 (p > 0.9999, n=4), and Grm8 (p=0.9947, n=4). H. Schematic representation of electrophysiological protocol stimulating glutamate release of Schaffer collaterals and recording field excitatory postsynaptic potentials (fEPSPs) from the stratum radiatum of CA1. Inset image highlights group III mGluR autoreceptor role in modulating glutamate release and subsequent postsynaptic response via activation of ionotropic glutamate receptors (iGluRs). I. Representative fEPSP traces for WT and Elfn2 KO mice. J. Quantitative analysis of fEPSP slope across various stimulations for WT and Elfn2 KO mice (10¼A, p > 0.9999, n=9; 20¼A, p=0.9991, n=9; 30¼A, p=0.6957, n=9; 40¼A, p=0.0487, n=9; 50¼A, p=0.0310, n=9; 60¼A, p=0.0227, n=9; 70¼A, p=0.0068, n=9; 80¼A, p=0.0092, n=9; 90¼A, p=0.0012, n=9). K. Quantitative analysis of fiber volley across various stimulations for WT and Elfn2 KO mice (10¼A-80 μA, p > 0.9999, n=9; 90¼A, p=0.9998, n=9). L. Linear regression comparing fEPSP slope to fiber volley in WT and Elfn2 KO mice (p
    Figure Legend Snippet: A. Schematic representation of generation of Elfn2 KO mouse replace exon2 on Elfn2 gene with LacZ-neo cassette for β-galactosidase reporter expression and disruption of ELFN2 protein expression (CDS, coding sequence; UTR, untranslated region). B. Immunohistochemistry for β-galactosidase expression (green) in Elfn2 KO mice coronal section and DAPI nuclear staining (blue). C. Western blot confirming lack of ELFN2 protein expression in Elfn2 KO mouse whole brain homogenate compared to wildtype mouse. GAPDH Western blot provided as loading control. D. Comparative body mass between wildtype and Elfn2 KO mice (p=0.1965, n=8). E. Comparative brain mass between wildtype and Elfn2 KO mice (p=0.5819, n=7). F. Western blotting and densitometric quantification of protein expression for various synaptic machineries compared between wildtype and Elfn2 KO mice: PSD-95 (p=0.1492, n=7), Synaptophysin (p=0.7168, n=7), GluA1 (p=0.6760, n=7), NMDAR 1 (p=0.3416, n=7), NMDAR 2B (p=0.8387, n=7), mGluR5 (p=0.1057, n=7), mGluR2 (p=0.3570, n=5–7), mGluR4 (p=0.0027, n=7), mGluR7 (p=0.0005, n=7), mGluR8 (p=0.0244, n=7), GABA A R α1 (p=0.3671, n=7). G. Quantitative real-time PCR for RNA expression of Elfn2 (p=0.0018, n=4), Grm2 (p > 0.9999, n=4), Grm5 (p > 0.9999, n=4), Grm4 (p=0.6416, n=4), Grm7 (p > 0.9999, n=4), and Grm8 (p=0.9947, n=4). H. Schematic representation of electrophysiological protocol stimulating glutamate release of Schaffer collaterals and recording field excitatory postsynaptic potentials (fEPSPs) from the stratum radiatum of CA1. Inset image highlights group III mGluR autoreceptor role in modulating glutamate release and subsequent postsynaptic response via activation of ionotropic glutamate receptors (iGluRs). I. Representative fEPSP traces for WT and Elfn2 KO mice. J. Quantitative analysis of fEPSP slope across various stimulations for WT and Elfn2 KO mice (10¼A, p > 0.9999, n=9; 20¼A, p=0.9991, n=9; 30¼A, p=0.6957, n=9; 40¼A, p=0.0487, n=9; 50¼A, p=0.0310, n=9; 60¼A, p=0.0227, n=9; 70¼A, p=0.0068, n=9; 80¼A, p=0.0092, n=9; 90¼A, p=0.0012, n=9). K. Quantitative analysis of fiber volley across various stimulations for WT and Elfn2 KO mice (10¼A-80 μA, p > 0.9999, n=9; 90¼A, p=0.9998, n=9). L. Linear regression comparing fEPSP slope to fiber volley in WT and Elfn2 KO mice (p

    Techniques Used: Expressing, Sequencing, Immunohistochemistry, Mouse Assay, Staining, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Activation Assay

    19) Product Images from "Spatial Training Ameliorates Long-Term Alzheimer’s Disease-Like Pathological Deficits by Reducing NLRP3 Inflammasomes in PR5 Mice"

    Article Title: Spatial Training Ameliorates Long-Term Alzheimer’s Disease-Like Pathological Deficits by Reducing NLRP3 Inflammasomes in PR5 Mice

    Journal: Neurotherapeutics

    doi: 10.1007/s13311-018-00698-w

    The NLRP3/caspase-1/IL-1β axis changes in PR5+NLRP3−/− mice after spatial training. ( a – d ) Expression of NLRP3, caspase-1, and IL-1β in the hippocampus after training. ( e , f ) The IL-1β and TNF-α levels in the hippocampus after training was detected by ELISA. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3 or n = 6). * p
    Figure Legend Snippet: The NLRP3/caspase-1/IL-1β axis changes in PR5+NLRP3−/− mice after spatial training. ( a – d ) Expression of NLRP3, caspase-1, and IL-1β in the hippocampus after training. ( e , f ) The IL-1β and TNF-α levels in the hippocampus after training was detected by ELISA. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3 or n = 6). * p

    Techniques Used: Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay

    Alteration of the NLRP3/caspase-1/IL-1β axis immediately after training and 3 months later. ( a – d ) Expression of NLRP3, caspase-1, and IL-1β in the hippocampus immediately after training. ( e – h ) Expression of NLRP3, caspase-1, and IL-1β in the hippocampus 3 months later. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3). ** p
    Figure Legend Snippet: Alteration of the NLRP3/caspase-1/IL-1β axis immediately after training and 3 months later. ( a – d ) Expression of NLRP3, caspase-1, and IL-1β in the hippocampus immediately after training. ( e – h ) Expression of NLRP3, caspase-1, and IL-1β in the hippocampus 3 months later. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3). ** p

    Techniques Used: Expressing

    Effects of spatial training on the expression of synapse-associated proteins and on the synaptic morphology in PR5+NLRP3+/− and PR5+NLRP3−/− mice. ( a – d ) Expression of presynaptic and postsynaptic proteins synaptophysin, PSD93, and PSD95 in the hippocampus after training. ( e – h ) The spine density in AO and BS dendrites in the hippocampus after training. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3 or n = 5). Scale bars, 1 μm. * p
    Figure Legend Snippet: Effects of spatial training on the expression of synapse-associated proteins and on the synaptic morphology in PR5+NLRP3+/− and PR5+NLRP3−/− mice. ( a – d ) Expression of presynaptic and postsynaptic proteins synaptophysin, PSD93, and PSD95 in the hippocampus after training. ( e – h ) The spine density in AO and BS dendrites in the hippocampus after training. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3 or n = 5). Scale bars, 1 μm. * p

    Techniques Used: Expressing, Mouse Assay

    Altered expression of Tau proteins after spatial training in the PR5+NLRP3+/− and PR5+NLRP3−/− mice. ( a – e ) The phosphorylation-dependent antibodies pT231, pS396, Tau-1, and Tau-5 as indicated measured the alteration of tau in different phosphorylation status after training in the PR5+NLRP3+/− and PR5+NLRP3−/− mice. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3). ** p
    Figure Legend Snippet: Altered expression of Tau proteins after spatial training in the PR5+NLRP3+/− and PR5+NLRP3−/− mice. ( a – e ) The phosphorylation-dependent antibodies pT231, pS396, Tau-1, and Tau-5 as indicated measured the alteration of tau in different phosphorylation status after training in the PR5+NLRP3+/− and PR5+NLRP3−/− mice. Blot images were cropped for comparison. GAPDH was used as a loading control in each sample. The values were represented as mean ± SEM ( n = 3). ** p

    Techniques Used: Expressing, Mouse Assay

    20) Product Images from "Heterogeneous nuclear ribonucleoprotein A1 regulates rhythmic synthesis of mouse Nfil3 protein via IRES-mediated translation"

    Article Title: Heterogeneous nuclear ribonucleoprotein A1 regulates rhythmic synthesis of mouse Nfil3 protein via IRES-mediated translation

    Journal: Scientific Reports

    doi: 10.1038/srep42882

    hnRNP A1 regulates Nfil3 protein but not RNA oscillation. ( a ) MC3T3-E1 cells were transfected with sh_M or sh_hnA1 expression vectors, incubated for 48 hours, treated with phenylephrine and harvested at the indicated time points. Samples were subjected to immunoblotting with the indicated antibodies. This result is representative of four independent experiments. Full-length blots are presented in Figure S10 and band of interest is indicated by a red box. ( b ) Relative sh_M (closed circles/solid line) and sh_hnA1 (open circles/dotted line) transfected Nfil3 protein were normalized to 14-3-3ζ protein, calculated and plotted. The ratio of sh_M transfected Nfil3/Gapdh protein at 12 hours after phenylephrine treatment was set to 1.0. Error bars represent mean ± SEM ( n = 4). ( c ) MC3T3-E1 cells prepared in panel ( a ) were used for RNA quantification. Reverse-transcription and quantitative real-time PCR were performed using specific primers. Relative sh_M (closed circles/solid line) and sh_hnA1 (open circles/dotted line) transfected Nfil3 mRNA were normalized to Gapdh mRNA. The sh_M transfected Nfil3 mRNA level at 12 hours after phenylephrine treatment was set to 1.0. To statistically analyze the oscillation between sh_M and sh_hnA1, a two-way ANOVA was performed. Error bars represent mean ± SEM ( n = 4). ns P ≥ 0.05.
    Figure Legend Snippet: hnRNP A1 regulates Nfil3 protein but not RNA oscillation. ( a ) MC3T3-E1 cells were transfected with sh_M or sh_hnA1 expression vectors, incubated for 48 hours, treated with phenylephrine and harvested at the indicated time points. Samples were subjected to immunoblotting with the indicated antibodies. This result is representative of four independent experiments. Full-length blots are presented in Figure S10 and band of interest is indicated by a red box. ( b ) Relative sh_M (closed circles/solid line) and sh_hnA1 (open circles/dotted line) transfected Nfil3 protein were normalized to 14-3-3ζ protein, calculated and plotted. The ratio of sh_M transfected Nfil3/Gapdh protein at 12 hours after phenylephrine treatment was set to 1.0. Error bars represent mean ± SEM ( n = 4). ( c ) MC3T3-E1 cells prepared in panel ( a ) were used for RNA quantification. Reverse-transcription and quantitative real-time PCR were performed using specific primers. Relative sh_M (closed circles/solid line) and sh_hnA1 (open circles/dotted line) transfected Nfil3 mRNA were normalized to Gapdh mRNA. The sh_M transfected Nfil3 mRNA level at 12 hours after phenylephrine treatment was set to 1.0. To statistically analyze the oscillation between sh_M and sh_hnA1, a two-way ANOVA was performed. Error bars represent mean ± SEM ( n = 4). ns P ≥ 0.05.

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

    21) Product Images from "A Nonsteroidal Novel Formulation Targeting Inflammatory and Pruritus-Related Mediators Modulates Experimental Allergic Contact Dermatitis"

    Article Title: A Nonsteroidal Novel Formulation Targeting Inflammatory and Pruritus-Related Mediators Modulates Experimental Allergic Contact Dermatitis

    Journal: Dermatology and Therapy

    doi: 10.1007/s13555-018-0223-8

    Novel formulation treatment attenuated inflammatory markers. a Sensitization of skin promoted upregulation of PAR-2, while novel formulation attenuated PAR-2 production. Ears treated with vehicle alone did not develop ACD, while DNFB sensitization (D) triggered upregulation of the itch marker PAR-2. Test substances attenuated the PAR-2 response by 22% and 69%. Typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). PAR-2 means + SEM, n = 7. * p = 0.002, # p = 0.016. b Skin sensitization promoted upregulation of TRPV4, while novel formulation prevented TRPV4 production. Similarly, ears treated with vehicle alone did not develop ACD; however, DNFB sensitization (D) triggered upregulation of the itch marker TRPV4. Test substance formulation B reduced TRPV4 formation to naïve control levels. Two typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). TRPV4 means + SEM, n = 7. * p = 0.007, # p = 0.026. c Skin sensitization induced an inflammatory response, while novel formulation blocked inflammation onset. DNFB sensitization triggered upregulation of the inflammatory marker TNFα, but formulation B prevented inflammation onset. Two typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). TNFα means + SEM, n = 7. * p = 0.249, # p = 0.210
    Figure Legend Snippet: Novel formulation treatment attenuated inflammatory markers. a Sensitization of skin promoted upregulation of PAR-2, while novel formulation attenuated PAR-2 production. Ears treated with vehicle alone did not develop ACD, while DNFB sensitization (D) triggered upregulation of the itch marker PAR-2. Test substances attenuated the PAR-2 response by 22% and 69%. Typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). PAR-2 means + SEM, n = 7. * p = 0.002, # p = 0.016. b Skin sensitization promoted upregulation of TRPV4, while novel formulation prevented TRPV4 production. Similarly, ears treated with vehicle alone did not develop ACD; however, DNFB sensitization (D) triggered upregulation of the itch marker TRPV4. Test substance formulation B reduced TRPV4 formation to naïve control levels. Two typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). TRPV4 means + SEM, n = 7. * p = 0.007, # p = 0.026. c Skin sensitization induced an inflammatory response, while novel formulation blocked inflammation onset. DNFB sensitization triggered upregulation of the inflammatory marker TNFα, but formulation B prevented inflammation onset. Two typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). TNFα means + SEM, n = 7. * p = 0.249, # p = 0.210

    Techniques Used: Marker, Western Blot

    22) Product Images from "Dysregulated post-synaptic density and endocytic zone in the amygdala of human heroin and cocaine abusers"

    Article Title: Dysregulated post-synaptic density and endocytic zone in the amygdala of human heroin and cocaine abusers

    Journal: Biological psychiatry

    doi: 10.1016/j.biopsych.2010.09.037

    Protein levels of glutamatergic markers in the lateral amygdala of human heroin and control subjects from the heroin abuse population (Study II). A. Representative WB images of GluA1 (106 kD), PSD-95 (95 kD), mGluR5 (130 kD), Homer 1b/c (45 kD), GluN1 (110) kD and GAPDH (38 kD) in two control and two heroin subjects. B. Comparison of the immunoreactivities between human heroin (n=27–28) and control subjects (n=8–13). Protein levels are presented as percent of mean control values (mean ± SEM). *, p
    Figure Legend Snippet: Protein levels of glutamatergic markers in the lateral amygdala of human heroin and control subjects from the heroin abuse population (Study II). A. Representative WB images of GluA1 (106 kD), PSD-95 (95 kD), mGluR5 (130 kD), Homer 1b/c (45 kD), GluN1 (110) kD and GAPDH (38 kD) in two control and two heroin subjects. B. Comparison of the immunoreactivities between human heroin (n=27–28) and control subjects (n=8–13). Protein levels are presented as percent of mean control values (mean ± SEM). *, p

    Techniques Used: Western Blot

    23) Product Images from "A translational silencing function of MCPIP1/Regnase-1 specified by the target site context"

    Article Title: A translational silencing function of MCPIP1/Regnase-1 specified by the target site context

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky106

    Increased TSE-mediated translational silencing in cells overexpressing MCPIP1. ( A ) HEK-293 cells were co-transfected with a GFP expression plasmid containing the TSE in its 3′ UTR (GFP-TSE) without (con) or with strep-tagged MCPIP1. Cell lysates were subjected to western blot in serial dilutions using antibodies against GFP or eEF2 as a loading control. ( B ) GFP-TSE mRNA was quantified by RT-qPCR (mean ± SD of quadruplicates from one out of three experiments with similar results). ( C ) HEK-293 cells were transfected with plasmids encoding GFP mRNAs without (GFP) or with the NFKBIZ TSE (GFP-TSE) together with empty vector (con) or strep-tagged MCPIP1 as indicated. Fluorescence intensity was determined by flow cytometry. ( D ) Mean fluorescence intensities (MFI,±SD of triplicates; n.s., not significant) of cells transfected with plasmids encoding GFP-TSE together with empty vector (con) or strep-tagged WT or RNase-deficient MCPIP1 (D141N). ( E ) Fold suppression of protein (determined as MFI) and mRNA amounts (determined by RT-qPCR and normalized to GAPDH mRNA) by the TSE or by strep-MCPIP1 expression or both was calculated by setting the values for GFP mRNA without TSE and in the absence of strep-MCPIP1 as 1 (mean ± SD, n = 5). Similar results were observed in two independent assays. ( F ) Cytoplasmic lysates from HEK-293 cells transfected with plasmids encoding β-globin (βG) or GFP mRNAs without or with the TSE and co-transfected with empty vector (con) or plasmids for strep-tagged WT or catalytically inactive MCPIP1 were fractionated on sucrose gradients. A typical result of adsorbance profiles at 254 nm for cells expressing empty vector (con) or strep-MCPIP1 is shown in the top panel. The amounts of mRNA in each fraction were determined by RT-qPCR and expressed in per cent of the sum detected in all fractions. Positions of ribosomal subunits, ribosomes and polysomes are indicated. The result is representative of three independent experiments.
    Figure Legend Snippet: Increased TSE-mediated translational silencing in cells overexpressing MCPIP1. ( A ) HEK-293 cells were co-transfected with a GFP expression plasmid containing the TSE in its 3′ UTR (GFP-TSE) without (con) or with strep-tagged MCPIP1. Cell lysates were subjected to western blot in serial dilutions using antibodies against GFP or eEF2 as a loading control. ( B ) GFP-TSE mRNA was quantified by RT-qPCR (mean ± SD of quadruplicates from one out of three experiments with similar results). ( C ) HEK-293 cells were transfected with plasmids encoding GFP mRNAs without (GFP) or with the NFKBIZ TSE (GFP-TSE) together with empty vector (con) or strep-tagged MCPIP1 as indicated. Fluorescence intensity was determined by flow cytometry. ( D ) Mean fluorescence intensities (MFI,±SD of triplicates; n.s., not significant) of cells transfected with plasmids encoding GFP-TSE together with empty vector (con) or strep-tagged WT or RNase-deficient MCPIP1 (D141N). ( E ) Fold suppression of protein (determined as MFI) and mRNA amounts (determined by RT-qPCR and normalized to GAPDH mRNA) by the TSE or by strep-MCPIP1 expression or both was calculated by setting the values for GFP mRNA without TSE and in the absence of strep-MCPIP1 as 1 (mean ± SD, n = 5). Similar results were observed in two independent assays. ( F ) Cytoplasmic lysates from HEK-293 cells transfected with plasmids encoding β-globin (βG) or GFP mRNAs without or with the TSE and co-transfected with empty vector (con) or plasmids for strep-tagged WT or catalytically inactive MCPIP1 were fractionated on sucrose gradients. A typical result of adsorbance profiles at 254 nm for cells expressing empty vector (con) or strep-MCPIP1 is shown in the top panel. The amounts of mRNA in each fraction were determined by RT-qPCR and expressed in per cent of the sum detected in all fractions. Positions of ribosomal subunits, ribosomes and polysomes are indicated. The result is representative of three independent experiments.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cytometry

    24) Product Images from "Microenvironmental Stiffness Enhances Glioma Cell Proliferation by Stimulating Epidermal Growth Factor Receptor Signaling"

    Article Title: Microenvironmental Stiffness Enhances Glioma Cell Proliferation by Stimulating Epidermal Growth Factor Receptor Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101771

    Microenvironmental stiffness regulates expression and phosphorylation of EGFR pathway components. The expression of activated EGFR, activated Akt and PI3K in U373-MG cells rises with increasing substrate stiffness (A). Similarly, the expression levels of EGFR and Akt in U373-MG cells rise with increasing substrate stiffness (B). Results represent quantification of at least three biological replicates on three separate Western blots, where the relative protein expression levels have been first normalized to the expression of GAPDH and then normalized to the expression level on the stiffest substrate of 119 kPa. Representative blots for each protein are on the right. *, P
    Figure Legend Snippet: Microenvironmental stiffness regulates expression and phosphorylation of EGFR pathway components. The expression of activated EGFR, activated Akt and PI3K in U373-MG cells rises with increasing substrate stiffness (A). Similarly, the expression levels of EGFR and Akt in U373-MG cells rise with increasing substrate stiffness (B). Results represent quantification of at least three biological replicates on three separate Western blots, where the relative protein expression levels have been first normalized to the expression of GAPDH and then normalized to the expression level on the stiffest substrate of 119 kPa. Representative blots for each protein are on the right. *, P

    Techniques Used: Expressing, Western Blot

    25) Product Images from "Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6-dependent survival pathways"

    Article Title: Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6-dependent survival pathways

    Journal: eLife

    doi: 10.7554/eLife.06132

    The effect of siRNA targeting SOX2 is specific. ( A ), PC9 cells were transfected with two different siRNA duplexes targeting SOX2 (or control siRNA), followed by addition of DMSO or 0.1 µM erlotinib for 24 hr and immunoblot of protein lysates with the indicated antibodies. ( B ), the degree of knockdown of SOX2 was quantitatively assessed by qPCR. Data are shown as mean Ct (normalized to GAPDH and untreated siCTRL cells) of 3 replicates −/+ SEM. DOI: http://dx.doi.org/10.7554/eLife.06132.032
    Figure Legend Snippet: The effect of siRNA targeting SOX2 is specific. ( A ), PC9 cells were transfected with two different siRNA duplexes targeting SOX2 (or control siRNA), followed by addition of DMSO or 0.1 µM erlotinib for 24 hr and immunoblot of protein lysates with the indicated antibodies. ( B ), the degree of knockdown of SOX2 was quantitatively assessed by qPCR. Data are shown as mean Ct (normalized to GAPDH and untreated siCTRL cells) of 3 replicates −/+ SEM. DOI: http://dx.doi.org/10.7554/eLife.06132.032

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction

    Quantitation of the effect of SOX2 knockdown on BIM levels. Each BIM isoform (EL, L, and S) was normalized to the GAPDH loading control and untreated, siCTRL cells. DOI: http://dx.doi.org/10.7554/eLife.06132.033
    Figure Legend Snippet: Quantitation of the effect of SOX2 knockdown on BIM levels. Each BIM isoform (EL, L, and S) was normalized to the GAPDH loading control and untreated, siCTRL cells. DOI: http://dx.doi.org/10.7554/eLife.06132.033

    Techniques Used: Quantitation Assay

    26) Product Images from "Tau Secretion and Propagation Is Regulated by p300/CBP via Autophagy-Lysosomal Pathway in Tauopathy"

    Article Title: Tau Secretion and Propagation Is Regulated by p300/CBP via Autophagy-Lysosomal Pathway in Tauopathy

    Journal: bioRxiv

    doi: 10.1101/418640

    p300 Impairs Autophagic Flux (A, B) p300 overexpression in HEK293T cells increases LC3 and p62 accumulation. (A) Representative immunoblot of LC3-I, -II, SQSMT/p62 and GAPDH in lysates of HEK293T cells transfected with tau alone or tau and p300 and serum-starved for 48 h. (B) Quantification of levels of LC3-II and p62 relative to GAPDH, normalized to control (tau alone). n=3 wells from three independent experiments. *p
    Figure Legend Snippet: p300 Impairs Autophagic Flux (A, B) p300 overexpression in HEK293T cells increases LC3 and p62 accumulation. (A) Representative immunoblot of LC3-I, -II, SQSMT/p62 and GAPDH in lysates of HEK293T cells transfected with tau alone or tau and p300 and serum-starved for 48 h. (B) Quantification of levels of LC3-II and p62 relative to GAPDH, normalized to control (tau alone). n=3 wells from three independent experiments. *p

    Techniques Used: Over Expression, Transfection

    37892 Promotes Autophagic Flux and Reduces Tau Secretion in Human iPSC-induced Neurons (Related to Figure 4 ) (A) Representative immunoblot of acH3K18, H3, ac-tau (K174), t-tau (HT7), and GAPDH in lysates of human iPSC-induced neurons treated with DMSO (control) or 37892 (50 μM) for 3 days. Quantification of levels of acH3K18 (B) and ac-tau (C) and t-tau (D) after 3 days of treatment, normalized to ctrl. (E) Quantification of relative tau secretion over 3 h, based on levels of extracellular tau and intracellular tau measured by ELISA and normalized to control. n=6 wells from three independent experiments. *p
    Figure Legend Snippet: 37892 Promotes Autophagic Flux and Reduces Tau Secretion in Human iPSC-induced Neurons (Related to Figure 4 ) (A) Representative immunoblot of acH3K18, H3, ac-tau (K174), t-tau (HT7), and GAPDH in lysates of human iPSC-induced neurons treated with DMSO (control) or 37892 (50 μM) for 3 days. Quantification of levels of acH3K18 (B) and ac-tau (C) and t-tau (D) after 3 days of treatment, normalized to ctrl. (E) Quantification of relative tau secretion over 3 h, based on levels of extracellular tau and intracellular tau measured by ELISA and normalized to control. n=6 wells from three independent experiments. *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    New p300 Inhibitor 37892 Reduces Tau Secretion (A)Diagram of the homogeneous time-resolved fluorescence assay. Step 1, the enzymatic reaction: p300 transfers an acetyl group from acetyl-CoA to tau, producing CoA and ac-tau. Step 2, detection: a rabbit ac-tau–specific antibody (mAB359) recognizes ac-tau, the europium cryptate-labeled anti-rabbit IgG-EuK (donor) binds to mAB359, and the anti-GST-D2 (acceptor) captures GST-tau. When tau is acetylated, the Eu donor and D2 acceptor are brought into proximity, allowing Forster resonance energy transfer (FRET) to occur. FRET is detected as a long-lived fluorescence signal at 665 nm. The fluorescence signal ratio 665/620 nm is proportional to the extent of ac-tau in the solution. (B)Work flow of the high-throughput screen. (C)Structure of SMDC37892. (D, E) 37892 inhibits p300 activity in HEK293T. (D) Representative immunoblots of acH3K18 and H3 in histone extracts of HEK293T cells treated with increasing doses of 37892 for 24 h. (E) Concentration-response curve for 37892, quantified from p300 activity in HEK293T cells. (F–J) 37892 treatment in hTau-expressing rat primary neurons. (F) Representative immunoblots of p62, LC3-I, -II and GAPDH in primary neurons treated with 37892 (50 μM) for 24 h. (G) Representative immunoblots of LC3-I, -II and actin in primary neurons treated with 37892 (50 μM), BafA1 (5 nM), or both, for 24 h. (H) Autophagic flux in control and 37892-treated neurons quantified from the increase in LC3-II levels in response to BafA1 treatment, normalized to control. (I) Quantification of intracellular tau levels by ELISA and normalized to control. (J) Quantification of relative tau secretion over 3 h, based on levels of extracellular tau and intracellular tau measured by ELISA and normalized to control. n=6 wells from three independent experiments. **p
    Figure Legend Snippet: New p300 Inhibitor 37892 Reduces Tau Secretion (A)Diagram of the homogeneous time-resolved fluorescence assay. Step 1, the enzymatic reaction: p300 transfers an acetyl group from acetyl-CoA to tau, producing CoA and ac-tau. Step 2, detection: a rabbit ac-tau–specific antibody (mAB359) recognizes ac-tau, the europium cryptate-labeled anti-rabbit IgG-EuK (donor) binds to mAB359, and the anti-GST-D2 (acceptor) captures GST-tau. When tau is acetylated, the Eu donor and D2 acceptor are brought into proximity, allowing Forster resonance energy transfer (FRET) to occur. FRET is detected as a long-lived fluorescence signal at 665 nm. The fluorescence signal ratio 665/620 nm is proportional to the extent of ac-tau in the solution. (B)Work flow of the high-throughput screen. (C)Structure of SMDC37892. (D, E) 37892 inhibits p300 activity in HEK293T. (D) Representative immunoblots of acH3K18 and H3 in histone extracts of HEK293T cells treated with increasing doses of 37892 for 24 h. (E) Concentration-response curve for 37892, quantified from p300 activity in HEK293T cells. (F–J) 37892 treatment in hTau-expressing rat primary neurons. (F) Representative immunoblots of p62, LC3-I, -II and GAPDH in primary neurons treated with 37892 (50 μM) for 24 h. (G) Representative immunoblots of LC3-I, -II and actin in primary neurons treated with 37892 (50 μM), BafA1 (5 nM), or both, for 24 h. (H) Autophagic flux in control and 37892-treated neurons quantified from the increase in LC3-II levels in response to BafA1 treatment, normalized to control. (I) Quantification of intracellular tau levels by ELISA and normalized to control. (J) Quantification of relative tau secretion over 3 h, based on levels of extracellular tau and intracellular tau measured by ELISA and normalized to control. n=6 wells from three independent experiments. **p

    Techniques Used: Fluorescence, Labeling, Förster Resonance Energy Transfer, High Throughput Screening Assay, Activity Assay, Western Blot, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

    p300 Promotes Tau Secretion by Reducing Autophagic Flux (A–D) Rapamycin treatment in HEK293T cells with and without p300 overexpression. (A) Representative immunoblots of LC3-I, LC3-II, p62, total tau (t-tau), phosphor-tau (p-tau, AT8), acetylated tau (ac-tau, K174), and GAPDH in lysate (5% of total) and conditioned medium (25% of total) of HEK293T cells transfected with tau alone or tau + p300. Serum-starved HEK293T cells were treated with DMSO (ctrl) or rapamycin (1 μM) for 24 h. The conditioned medium was concentrated 25-fold. (B, C, D) Quantification of t-tau secretion (B) and p-tau secretion (C) relative to intracellular levels and normalized to control. n=3 wells from three independent experiments. * , # p
    Figure Legend Snippet: p300 Promotes Tau Secretion by Reducing Autophagic Flux (A–D) Rapamycin treatment in HEK293T cells with and without p300 overexpression. (A) Representative immunoblots of LC3-I, LC3-II, p62, total tau (t-tau), phosphor-tau (p-tau, AT8), acetylated tau (ac-tau, K174), and GAPDH in lysate (5% of total) and conditioned medium (25% of total) of HEK293T cells transfected with tau alone or tau + p300. Serum-starved HEK293T cells were treated with DMSO (ctrl) or rapamycin (1 μM) for 24 h. The conditioned medium was concentrated 25-fold. (B, C, D) Quantification of t-tau secretion (B) and p-tau secretion (C) relative to intracellular levels and normalized to control. n=3 wells from three independent experiments. * , # p

    Techniques Used: Over Expression, Western Blot, Transfection

    27) Product Images from "NEDD9/Arf6-Dependent Endocytic Trafficking of Matrix Metalloproteinase 14: A Novel Mechanism for Blocking Mesenchymal Cell Invasion and Metastasis of Breast Cancer"

    Article Title: NEDD9/Arf6-Dependent Endocytic Trafficking of Matrix Metalloproteinase 14: A Novel Mechanism for Blocking Mesenchymal Cell Invasion and Metastasis of Breast Cancer

    Journal: Oncogene

    doi: 10.1038/onc.2014.297

    NEDD9 regulates inactivation of Arf6 GTPase (a) Representative image of WB analysis with anti-Arf6. Top: pull-down of Arf6-GTP from shControl (Con) and shNEDD9 (N1, N2) MDA-MB-231 cells; Bottom: whole cell lysate (WCL) used for pull-down, WB with anti-Arf6, -NEDD9 (2G9), -GAPDH antibodies. (b) Quantification of relative intensity units (RIU) of WBs from (a), Arf6-total in WCL and Arf6-GTP in pull-down); three independent experiments; graphs are mean RIU as % to control (Con, assigned as 100%) ±S.E.M. one-way ANOVA with Dunnett's post-hoc analysis Arf6 total: *p=0.0013,
    Figure Legend Snippet: NEDD9 regulates inactivation of Arf6 GTPase (a) Representative image of WB analysis with anti-Arf6. Top: pull-down of Arf6-GTP from shControl (Con) and shNEDD9 (N1, N2) MDA-MB-231 cells; Bottom: whole cell lysate (WCL) used for pull-down, WB with anti-Arf6, -NEDD9 (2G9), -GAPDH antibodies. (b) Quantification of relative intensity units (RIU) of WBs from (a), Arf6-total in WCL and Arf6-GTP in pull-down); three independent experiments; graphs are mean RIU as % to control (Con, assigned as 100%) ±S.E.M. one-way ANOVA with Dunnett's post-hoc analysis Arf6 total: *p=0.0013,

    Techniques Used: Western Blot, Multiple Displacement Amplification

    NEDD9 depletion leads to redistribution of MMP14 to the cell surface (a-b) WB analysis of WCL from MDA-MB-231 (a) and Hs578T (b) cells expressing siRNAs against NEDD9 (N1, N2) or non-targeting control siRNA (Con) with anti-NEDD9 (2G9), -MMP14, -GAPDH antibodies. (c-d) MMP14-specific fluorogenic substrate degradation assay in cells as in (a-b). Fluorescence was measured at 328/400 nm excitation/emission. Data plotted as % of RFU to shControl ±S.E.M; n=3; one-way ANOVA with Dunnett's post-hoc analysis for MDA-MB-231: p
    Figure Legend Snippet: NEDD9 depletion leads to redistribution of MMP14 to the cell surface (a-b) WB analysis of WCL from MDA-MB-231 (a) and Hs578T (b) cells expressing siRNAs against NEDD9 (N1, N2) or non-targeting control siRNA (Con) with anti-NEDD9 (2G9), -MMP14, -GAPDH antibodies. (c-d) MMP14-specific fluorogenic substrate degradation assay in cells as in (a-b). Fluorescence was measured at 328/400 nm excitation/emission. Data plotted as % of RFU to shControl ±S.E.M; n=3; one-way ANOVA with Dunnett's post-hoc analysis for MDA-MB-231: p

    Techniques Used: Western Blot, Multiple Displacement Amplification, Expressing, Degradation Assay, Fluorescence

    28) Product Images from "ShcA promotes chondrocyte hypertrophic commitment and osteoarthritis in mice through RunX2 nuclear translocation and YAP1 inactivation"

    Article Title: ShcA promotes chondrocyte hypertrophic commitment and osteoarthritis in mice through RunX2 nuclear translocation and YAP1 inactivation

    Journal: bioRxiv

    doi: 10.1101/2020.07.16.206870

    Decreased ERKl/2 and RunX2 activation and increased YAP1 activation in TwShcA- mice and ShcA deficient cells. A) Western blot analysis and relative quantification of phospho-ERK1/2, nuclear and cytosolic RunX2, phospho-YAP1, YAP1, ShcA and GAPDH proteins levels in knee joint articular chondrocytes isolated from mice that express (TwShcA+) or lack ShcA in chondrocytes (TwShcA-) (n= 8 mice in each group for phospho-ERK1/2 ShcA, n= 5 mice in each group for RunX2, phospho-YAP1, YAP1). B) phospho-ERK1/2 staining of tibial growth plate in mice that express (TwShcA+) or lack ShcA in chondrocytes (TwShcA-). Scale bars 100 μm. C) Representative confocal immunostaining of RunX2 (upper panel) and YAP1 (lower panel) in articular chondrocytes isolated from mice that express (TwShcA+) or lack ShcA in chondrocytes (TwShcA-) (n= 3 separate experiments). Scale bars 10 μm. D) Western-blot analysis and relative quantification of nuclear and cytosolic YAP1 protein levels in ATDC5 cells down-regulated for ShcA (siShcA) and control cells (siCTRL) (n= 4 experiments in each group). *p
    Figure Legend Snippet: Decreased ERKl/2 and RunX2 activation and increased YAP1 activation in TwShcA- mice and ShcA deficient cells. A) Western blot analysis and relative quantification of phospho-ERK1/2, nuclear and cytosolic RunX2, phospho-YAP1, YAP1, ShcA and GAPDH proteins levels in knee joint articular chondrocytes isolated from mice that express (TwShcA+) or lack ShcA in chondrocytes (TwShcA-) (n= 8 mice in each group for phospho-ERK1/2 ShcA, n= 5 mice in each group for RunX2, phospho-YAP1, YAP1). B) phospho-ERK1/2 staining of tibial growth plate in mice that express (TwShcA+) or lack ShcA in chondrocytes (TwShcA-). Scale bars 100 μm. C) Representative confocal immunostaining of RunX2 (upper panel) and YAP1 (lower panel) in articular chondrocytes isolated from mice that express (TwShcA+) or lack ShcA in chondrocytes (TwShcA-) (n= 3 separate experiments). Scale bars 10 μm. D) Western-blot analysis and relative quantification of nuclear and cytosolic YAP1 protein levels in ATDC5 cells down-regulated for ShcA (siShcA) and control cells (siCTRL) (n= 4 experiments in each group). *p

    Techniques Used: Activation Assay, Mouse Assay, Western Blot, Isolation, Staining, Immunostaining

    29) Product Images from "Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer"

    Article Title: Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-017-1327-0

    Identification of transgenic piglets. a Obtained partly P53 KO piglets. b The sequences of the P53 mutation in cloned piglets. The WT sequence is shown above. Deletions (denoted with “Δ” and the number of base pairs) are identified. c The relative expression levels of P53 mRNA in the different tissues from P53 KO and WT piglets. The relative expression levels of P53 mRNA in brain, muscle, kidney, heart, liver and kidney tissues of P53 KO and WT piglets were measured using q-PCR. Expression of the GAPDH gene was used to normalize the values of P53 . * p
    Figure Legend Snippet: Identification of transgenic piglets. a Obtained partly P53 KO piglets. b The sequences of the P53 mutation in cloned piglets. The WT sequence is shown above. Deletions (denoted with “Δ” and the number of base pairs) are identified. c The relative expression levels of P53 mRNA in the different tissues from P53 KO and WT piglets. The relative expression levels of P53 mRNA in brain, muscle, kidney, heart, liver and kidney tissues of P53 KO and WT piglets were measured using q-PCR. Expression of the GAPDH gene was used to normalize the values of P53 . * p

    Techniques Used: Transgenic Assay, Mutagenesis, Clone Assay, Sequencing, Expressing, Polymerase Chain Reaction

    Phenotype detections. a The intracellular localization of P53 was analyzed using confocal fluorescence microscopy. The fibroblast cells from P53 KO piglets and WT piglets were treated with DOX at 100 µM for 24 h and stained with Hoechst 33342 (blue) and an anti-P53 antibody (green). b The fibroblast cells were treated as described above and protein expression levels were examined by Western blotting. P53 and P21 protein expression in the treated fibroblasts are shown in cropped blots using an anti-P53 and anti-P21 monoclonal antibody. Anti-GAPDH served as a loading control
    Figure Legend Snippet: Phenotype detections. a The intracellular localization of P53 was analyzed using confocal fluorescence microscopy. The fibroblast cells from P53 KO piglets and WT piglets were treated with DOX at 100 µM for 24 h and stained with Hoechst 33342 (blue) and an anti-P53 antibody (green). b The fibroblast cells were treated as described above and protein expression levels were examined by Western blotting. P53 and P21 protein expression in the treated fibroblasts are shown in cropped blots using an anti-P53 and anti-P21 monoclonal antibody. Anti-GAPDH served as a loading control

    Techniques Used: Fluorescence, Microscopy, Staining, Expressing, Western Blot

    30) Product Images from "Cathepsin D is the main lysosomal enzyme involved in the degradation of ?-synuclein and generation of its carboxy-terminally truncated species †"

    Article Title: Cathepsin D is the main lysosomal enzyme involved in the degradation of ?-synuclein and generation of its carboxy-terminally truncated species †

    Journal: Biochemistry

    doi: 10.1021/bi800699v

    Effect of calpain I reduction on α-syn levels in 3D5 cells A . 3D5 cells were infected with 1/2 and 1/10 dilutions of viral-medium containing a non-target control (NT) or constructs 31, 31, 33, and 34 targeting calpain I RNA. Three days after infection, cell lysates were prepared and 10 μg from each sample were analyzed by Western blot. The blot was probed with antibodies against calpain I, α-syn (syn-1), GAPDH, and ß-actin. B . Quantitation of calpain I and α-syn in 3D5 lysates expressing constructs targeting calpain I was done by scanning the X-ray films and using ImageQuant software. The values were normalized to GAPDH, and expressed as a percentage of the NT value. The percentages were as follows: construct 31, 26% (n=5); 32, 22% (n=7); 33, 22% (n=7); and 34, 7% (n=8). Similar values were obtained when ß-actin was used for the calculations. The broken line in the plot indicates 100%. *=P
    Figure Legend Snippet: Effect of calpain I reduction on α-syn levels in 3D5 cells A . 3D5 cells were infected with 1/2 and 1/10 dilutions of viral-medium containing a non-target control (NT) or constructs 31, 31, 33, and 34 targeting calpain I RNA. Three days after infection, cell lysates were prepared and 10 μg from each sample were analyzed by Western blot. The blot was probed with antibodies against calpain I, α-syn (syn-1), GAPDH, and ß-actin. B . Quantitation of calpain I and α-syn in 3D5 lysates expressing constructs targeting calpain I was done by scanning the X-ray films and using ImageQuant software. The values were normalized to GAPDH, and expressed as a percentage of the NT value. The percentages were as follows: construct 31, 26% (n=5); 32, 22% (n=7); 33, 22% (n=7); and 34, 7% (n=8). Similar values were obtained when ß-actin was used for the calculations. The broken line in the plot indicates 100%. *=P

    Techniques Used: Infection, Construct, Western Blot, Quantitation Assay, Expressing, Software

    31) Product Images from "Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value"

    Article Title: Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value

    Journal: Leukemia

    doi: 10.1038/leu.2014.100

    Generation and characterization of the anti-mutated CALR antibody. In panel a , the sequence of the 17-mer peptide used for generation of the anti-mutated CALR antibody is shown in relation with the amino-acid sequence of wild-type CALR and the predicted sequence of mutated CALR originated from the del52 and ins5 abnormalities. In panel b , gel electrophoresis of the mutated calreticulin prepared in BL21DE3RIPL (lanes 2 and 3) and JM109DE3 (lanes 4 and 5) E. Coli strains; only in the latter strain successful production of calreticulin was obtained. Calreticulin has a molecular weight of 47 kDa, but migrates in these conditions at an apparent higher molecular weight likely owing to a specific conformational pattern. In panel c , western blot analysis shows anti-mutated CALR antibody selectivity for mutated protein vs the wild-type form. CALR recombinant protein expressed in E. Coli was used as a positive control. GAPDH was used as a loading control.
    Figure Legend Snippet: Generation and characterization of the anti-mutated CALR antibody. In panel a , the sequence of the 17-mer peptide used for generation of the anti-mutated CALR antibody is shown in relation with the amino-acid sequence of wild-type CALR and the predicted sequence of mutated CALR originated from the del52 and ins5 abnormalities. In panel b , gel electrophoresis of the mutated calreticulin prepared in BL21DE3RIPL (lanes 2 and 3) and JM109DE3 (lanes 4 and 5) E. Coli strains; only in the latter strain successful production of calreticulin was obtained. Calreticulin has a molecular weight of 47 kDa, but migrates in these conditions at an apparent higher molecular weight likely owing to a specific conformational pattern. In panel c , western blot analysis shows anti-mutated CALR antibody selectivity for mutated protein vs the wild-type form. CALR recombinant protein expressed in E. Coli was used as a positive control. GAPDH was used as a loading control.

    Techniques Used: Sequencing, Nucleic Acid Electrophoresis, Molecular Weight, Western Blot, Recombinant, Positive Control

    32) Product Images from "Semaphorin 4D Promotes Skeletal Metastasis in Breast Cancer"

    Article Title: Semaphorin 4D Promotes Skeletal Metastasis in Breast Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150151

    Sema4D produced by breast cancer cells inhibits mineralization and promotes RhoA-mediated production of IL-8 by osteoblasts. A. Breast cancer cell line MDA-MB-231 expresses high levels of Sema4D in an immunoblot compared to the non-tumorigenic line MCF-12 and T47D, which rarely metastasize to bone (top panel). GAPDH was used as the loading control (lower panel). B. MDA-MB-231 cells infected with lentivirus coding for a scrambled shRNA (C) express Sema4D in whole cell lysates, levels of which are greatly reduced in cells infected with lentivirus expressing Sema4D shRNA (top left panel). MCF-12A cells, which express very little endogenous Sema4D (C), are induced to overexpress it through lentiviral-mediated transfer of the wild-type construct (Sema4D, top right panel). GAPDH was used as a loading control (middle panel). sSema4D is present in media conditioned by control MDA-MB-231 cells and MCF-12A overexpressing Sema4D, but not in MDA-MB-231 with silenced Sema4D or control MCF-12A infected with a lentivirus containing an empty vector (bottom panel). C. MC3T3-mediated matrix deposition and mineralization, shown by Alizarin Red stain, is inhibited by media conditioned by control MDA-MB-231 (C, first column, left panels), but not in those where Sema4D is silenced by shRNA (Sema4D shRNA, second column, left). When Plexin-B1 is silenced in MC3T3 (PB1 shRNA, second row), or anti-Sema4D blocking antibody is administered (bottom row), mineralization is restored. Media conditioned by control MCF-12A had no effect on mineralization (C, first column, right panels) unless these cells were over-expressing Sema4D, in which case there was inhibition (Sema4D, second column, right). Mineralization was restored when Plexin-B1 was silenced in osteoblasts (PB1 shRNA, second row) or blocking antibody was present (bottom row). Isotype matched IgG was used as a control for both experimental groups. D. Quantification of results by absorbance at 405 nm (Y-axis) of the results shown in (B) from three independent experiments. MDA-MB-231 cells, left bar graph; MCF-12A, right. E. Incubation of the human osteoblast line HOB with media conditioned by MDA-MB-231 induces production of IL-8 (concentration in ng/ml, Y-axis), unless Sema4D is silenced in MDA-MB-231 by shRNA or Plexin-B1 signaling to RhoA is inhibited by addition of the Clostridium botulinum toxin C3 (*, p ≤ 0.05; **, p ≤ 0.01).
    Figure Legend Snippet: Sema4D produced by breast cancer cells inhibits mineralization and promotes RhoA-mediated production of IL-8 by osteoblasts. A. Breast cancer cell line MDA-MB-231 expresses high levels of Sema4D in an immunoblot compared to the non-tumorigenic line MCF-12 and T47D, which rarely metastasize to bone (top panel). GAPDH was used as the loading control (lower panel). B. MDA-MB-231 cells infected with lentivirus coding for a scrambled shRNA (C) express Sema4D in whole cell lysates, levels of which are greatly reduced in cells infected with lentivirus expressing Sema4D shRNA (top left panel). MCF-12A cells, which express very little endogenous Sema4D (C), are induced to overexpress it through lentiviral-mediated transfer of the wild-type construct (Sema4D, top right panel). GAPDH was used as a loading control (middle panel). sSema4D is present in media conditioned by control MDA-MB-231 cells and MCF-12A overexpressing Sema4D, but not in MDA-MB-231 with silenced Sema4D or control MCF-12A infected with a lentivirus containing an empty vector (bottom panel). C. MC3T3-mediated matrix deposition and mineralization, shown by Alizarin Red stain, is inhibited by media conditioned by control MDA-MB-231 (C, first column, left panels), but not in those where Sema4D is silenced by shRNA (Sema4D shRNA, second column, left). When Plexin-B1 is silenced in MC3T3 (PB1 shRNA, second row), or anti-Sema4D blocking antibody is administered (bottom row), mineralization is restored. Media conditioned by control MCF-12A had no effect on mineralization (C, first column, right panels) unless these cells were over-expressing Sema4D, in which case there was inhibition (Sema4D, second column, right). Mineralization was restored when Plexin-B1 was silenced in osteoblasts (PB1 shRNA, second row) or blocking antibody was present (bottom row). Isotype matched IgG was used as a control for both experimental groups. D. Quantification of results by absorbance at 405 nm (Y-axis) of the results shown in (B) from three independent experiments. MDA-MB-231 cells, left bar graph; MCF-12A, right. E. Incubation of the human osteoblast line HOB with media conditioned by MDA-MB-231 induces production of IL-8 (concentration in ng/ml, Y-axis), unless Sema4D is silenced in MDA-MB-231 by shRNA or Plexin-B1 signaling to RhoA is inhibited by addition of the Clostridium botulinum toxin C3 (*, p ≤ 0.05; **, p ≤ 0.01).

    Techniques Used: Produced, Multiple Displacement Amplification, Infection, shRNA, Expressing, Construct, Plasmid Preparation, Staining, Blocking Assay, Inhibition, Incubation, Concentration Assay

    33) Product Images from "Annexin Peptide Ac2-26 Suppresses TNF?-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells"

    Article Title: Annexin Peptide Ac2-26 Suppresses TNF?-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060790

    The stimulatory effect of tumour necrosis factor (TNFα) on adhesion molecule gene upregulation requires Rac1 in HMECs. (A) TNFα (2–50 ng/ml) reduced Nox4 gene expression (B) and pretreatment of Ac2-26 (0.5 µM) did not affect the TNFα mediated mRNA downregulation. TNFα (2–50 ng/ml) alone (C) and in combination of Ac2-26 (0.5 µM) (D) did not affect Nox2 mRNA expression. TNFα (20 ng/ml) stimulated ICAM-1 mRNA (E) and protein (G) and VCAM-1 mRNA (F) and protein (G) upregulation was blunted by Ac2-26 (0.5 µM) and diphenyleneiodonium (DPI, 1 µM) in cells trasnfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1 and VCAM-1 upregulation and this inhibition was not potentiated by pretreatment of Ac2-26 and DPI. mRNA expression was normalized to control with TNFα stimulation in cells transfected with GFP. GAPDH was used to confirm equal loading. Data are mean ± SEM, n = 5 to 8. * P
    Figure Legend Snippet: The stimulatory effect of tumour necrosis factor (TNFα) on adhesion molecule gene upregulation requires Rac1 in HMECs. (A) TNFα (2–50 ng/ml) reduced Nox4 gene expression (B) and pretreatment of Ac2-26 (0.5 µM) did not affect the TNFα mediated mRNA downregulation. TNFα (2–50 ng/ml) alone (C) and in combination of Ac2-26 (0.5 µM) (D) did not affect Nox2 mRNA expression. TNFα (20 ng/ml) stimulated ICAM-1 mRNA (E) and protein (G) and VCAM-1 mRNA (F) and protein (G) upregulation was blunted by Ac2-26 (0.5 µM) and diphenyleneiodonium (DPI, 1 µM) in cells trasnfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1 and VCAM-1 upregulation and this inhibition was not potentiated by pretreatment of Ac2-26 and DPI. mRNA expression was normalized to control with TNFα stimulation in cells transfected with GFP. GAPDH was used to confirm equal loading. Data are mean ± SEM, n = 5 to 8. * P

    Techniques Used: Expressing, Plasmid Preparation, Dominant Negative Mutation, Inhibition, Transfection

    The stimulatory effect of tumour necrosis factor (TNFα) is NADPH oxidase dependent in HMECs. (A) Inhibitors of other enzymatic sources of ROS, allopurinol (Allo, 100 µM), indomethacin (Indo, 3 µM), L-NAME (100 µM) and rotenone (Rote, 1 µM) did not affect TNFα-induced superoxide generation, but it was inhibited by the Nox inhibitor diphenyleneiodonium (DPI, 1 µM). Cells were treated with TNFα (20 ng/ml) for 24 h prior to incubation with inhibitors, which were incubated with HMECs for 30 min prior to measurement of superoxide. (B, C) TNFα (20 ng/ml) stimulated ICAM-1 and VCAM-1 mRNA upregulation in cells transfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1and VCAM-1 mRNA upregulation. mRNA expression was normalized to control with TNFα stimulation in cells transfected with GFP. (D) TNFα (20 ng/ml) stimulated ICAM-1 and VCAM-1 mRNA upregulation in cells transfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1and VCAM-1 protein expression. GAPDH was used as a reference. (E,F) DPI (1 µM) suppressed the stimulatory effect of TNFα (20 ng/ml) on mRNA expression of ICAM-1 and VCAM-1 respectively. mRNA expression was expressed as fold change and normalized to control (Ctrl) with TNFα stimulation. (G) DPI (1 µM) suppressed the stimulatory effect of TNFα (20 ng/ml) on protein expression of ICAM-1 and VCAM-1. GAPDH was used to confirm equal loading. Data are mean ± SEM ; n = 3. * P
    Figure Legend Snippet: The stimulatory effect of tumour necrosis factor (TNFα) is NADPH oxidase dependent in HMECs. (A) Inhibitors of other enzymatic sources of ROS, allopurinol (Allo, 100 µM), indomethacin (Indo, 3 µM), L-NAME (100 µM) and rotenone (Rote, 1 µM) did not affect TNFα-induced superoxide generation, but it was inhibited by the Nox inhibitor diphenyleneiodonium (DPI, 1 µM). Cells were treated with TNFα (20 ng/ml) for 24 h prior to incubation with inhibitors, which were incubated with HMECs for 30 min prior to measurement of superoxide. (B, C) TNFα (20 ng/ml) stimulated ICAM-1 and VCAM-1 mRNA upregulation in cells transfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1and VCAM-1 mRNA upregulation. mRNA expression was normalized to control with TNFα stimulation in cells transfected with GFP. (D) TNFα (20 ng/ml) stimulated ICAM-1 and VCAM-1 mRNA upregulation in cells transfected with control GFP plasmid. Dominant negative Rac1 (N17Rac1) reduced TNFα-mediated ICAM-1and VCAM-1 protein expression. GAPDH was used as a reference. (E,F) DPI (1 µM) suppressed the stimulatory effect of TNFα (20 ng/ml) on mRNA expression of ICAM-1 and VCAM-1 respectively. mRNA expression was expressed as fold change and normalized to control (Ctrl) with TNFα stimulation. (G) DPI (1 µM) suppressed the stimulatory effect of TNFα (20 ng/ml) on protein expression of ICAM-1 and VCAM-1. GAPDH was used to confirm equal loading. Data are mean ± SEM ; n = 3. * P

    Techniques Used: Incubation, Transfection, Plasmid Preparation, Dominant Negative Mutation, Expressing

    34) Product Images from "The Simian Virus 40 Late Viral Protein VP4 Disrupts the Nuclear Envelope for Viral Release"

    Article Title: The Simian Virus 40 Late Viral Protein VP4 Disrupts the Nuclear Envelope for Viral Release

    Journal: Journal of Virology

    doi: 10.1128/JVI.07047-11

    VP4 is concentrated along the nuclear envelope. (A) Cos7 cells were transiently transfected with either empty vector (−) or VP4 (+) in the presence of the tetracycline repressor. Tetracycline was added for the indicated times. Whole-cell lysates were immunoblotted with antisera against myc to detect VP4 or the loading control GAPDH. (B) Confocal immunofluorescence microscopy localization of VP4 in Cos7 cells was determined after 6 h of induction. Samples were labeled with myc (VP4-myc), ERp57, emerin, or lamin antisera and stained with either Hoechst or DAPI (DAPI, 4′,6-diamidino-2-phenylindole) as indicated. A fraction of the VP4-expressing cells displayed profound nuclear envelope disruption, as displayed in the lamin lower panel. Bars, 10 μm.
    Figure Legend Snippet: VP4 is concentrated along the nuclear envelope. (A) Cos7 cells were transiently transfected with either empty vector (−) or VP4 (+) in the presence of the tetracycline repressor. Tetracycline was added for the indicated times. Whole-cell lysates were immunoblotted with antisera against myc to detect VP4 or the loading control GAPDH. (B) Confocal immunofluorescence microscopy localization of VP4 in Cos7 cells was determined after 6 h of induction. Samples were labeled with myc (VP4-myc), ERp57, emerin, or lamin antisera and stained with either Hoechst or DAPI (DAPI, 4′,6-diamidino-2-phenylindole) as indicated. A fraction of the VP4-expressing cells displayed profound nuclear envelope disruption, as displayed in the lamin lower panel. Bars, 10 μm.

    Techniques Used: Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Labeling, Staining, Expressing

    35) Product Images from "Oncogenic potential of N-terminal deletion and S45Y mutant β-catenin in promoting hepatocellular carcinoma development in mice"

    Article Title: Oncogenic potential of N-terminal deletion and S45Y mutant β-catenin in promoting hepatocellular carcinoma development in mice

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4870-z

    Biochemical analysis of HCC lesions of c-Met/β-cateninS45Y c-Met/∆N90-β-catenin mice. Western blot analysis of c-Met, p-Met, β-catenin, Myc tag, p-β-catenin (S45), p-β-catenin (S33/S37/T41), p-β-catenin (S675), p-β-catenin (S552), GS, and cyclin D1 in normal liver as well as HCC lesions from c-Met/β-cateninS45Y c-Met/∆N90-β-catenin mice. GAPDH were used as a loading control. Please note the presence of two bands for the β-catenin protein. The upper band represents endogenous β-catenin protein or β-cateninS45Y protein. The lower band represents ectopically injected ∆N90-β-catenin protein
    Figure Legend Snippet: Biochemical analysis of HCC lesions of c-Met/β-cateninS45Y c-Met/∆N90-β-catenin mice. Western blot analysis of c-Met, p-Met, β-catenin, Myc tag, p-β-catenin (S45), p-β-catenin (S33/S37/T41), p-β-catenin (S675), p-β-catenin (S552), GS, and cyclin D1 in normal liver as well as HCC lesions from c-Met/β-cateninS45Y c-Met/∆N90-β-catenin mice. GAPDH were used as a loading control. Please note the presence of two bands for the β-catenin protein. The upper band represents endogenous β-catenin protein or β-cateninS45Y protein. The lower band represents ectopically injected ∆N90-β-catenin protein

    Techniques Used: Mouse Assay, Western Blot, Injection

    36) Product Images from "miR-93-5p-Containing Exosomes Treatment Attenuates Acute Myocardial Infarction-Induced Myocardial Damage"

    Article Title: miR-93-5p-Containing Exosomes Treatment Attenuates Acute Myocardial Infarction-Induced Myocardial Damage

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.01.010

    Both Atg7 and TLR4 Were Direct Targets of miR-93-5p (A) Complementary sequences between miR-93-5p and the 3′ UTR of Atg7 mRNA were obtained using publicly available algorithms. The mutated version of the Atg7 3′ UTR is also shown. (B) The 3′ UTR of Atg7 was fused to the luciferase coding region (PYr-Atg7 3′ UTR) and co-transfected into HEK293T cells with miR-93-5p mimic to confirm that Atg7 is the target of miR-93-5p. The PYr-RGS-17 3′ UTR and miR-93-5p mimic constructs were co-transfected into HEK293T cells with a control vector, and the relative luciferase activity was determined 48 hr after transfection. Data are expressed as the mean ± SD. (C and D) Western blotting (C) and RT-PCR (D) analysis of Atg7 expression in H9c2 cells after transfection with miR-93-5p mimic (n = 3). GAPDH expression levels were measured as an endogenous control. The data are expressed as the mean ± SD. (E) Complementary sequences between miR-93-5p and the 3′ UTR of TLR4. The mutated version of the TLR4 3′ UTR is also shown. (F) The 3′ UTR of TLR4 was fused to the luciferase coding region (PYr-TLR4 3′ UTR) and co-transfected into HEK293T cells with miR-93-5p mimic to confirm that TLR4 is the target of miR-93-5p. The PYr-RGS-17 3′ UTR and miR-93-5p mimic constructs were co-transfected into HEK293T cells with a control vector, and the relative luciferase activity was determined 48 hr after transfection. The data are expressed as mean ± SD. (G and H) Western blotting (G) and RT-PCR (H) analysis of TLR4 expression in H9c2 cells after transfection with miR-93-5p mimic (n = 3). GAPDH expression levels were measured as an endogenous control. The data are expressed as mean ± SD. ***p
    Figure Legend Snippet: Both Atg7 and TLR4 Were Direct Targets of miR-93-5p (A) Complementary sequences between miR-93-5p and the 3′ UTR of Atg7 mRNA were obtained using publicly available algorithms. The mutated version of the Atg7 3′ UTR is also shown. (B) The 3′ UTR of Atg7 was fused to the luciferase coding region (PYr-Atg7 3′ UTR) and co-transfected into HEK293T cells with miR-93-5p mimic to confirm that Atg7 is the target of miR-93-5p. The PYr-RGS-17 3′ UTR and miR-93-5p mimic constructs were co-transfected into HEK293T cells with a control vector, and the relative luciferase activity was determined 48 hr after transfection. Data are expressed as the mean ± SD. (C and D) Western blotting (C) and RT-PCR (D) analysis of Atg7 expression in H9c2 cells after transfection with miR-93-5p mimic (n = 3). GAPDH expression levels were measured as an endogenous control. The data are expressed as the mean ± SD. (E) Complementary sequences between miR-93-5p and the 3′ UTR of TLR4. The mutated version of the TLR4 3′ UTR is also shown. (F) The 3′ UTR of TLR4 was fused to the luciferase coding region (PYr-TLR4 3′ UTR) and co-transfected into HEK293T cells with miR-93-5p mimic to confirm that TLR4 is the target of miR-93-5p. The PYr-RGS-17 3′ UTR and miR-93-5p mimic constructs were co-transfected into HEK293T cells with a control vector, and the relative luciferase activity was determined 48 hr after transfection. The data are expressed as mean ± SD. (G and H) Western blotting (G) and RT-PCR (H) analysis of TLR4 expression in H9c2 cells after transfection with miR-93-5p mimic (n = 3). GAPDH expression levels were measured as an endogenous control. The data are expressed as mean ± SD. ***p

    Techniques Used: Luciferase, Transfection, Construct, Plasmid Preparation, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

    37) Product Images from "Loss of Caveolin 1 is Associated With the Expression of Aquaporin 1 and Bladder Dysfunction in Mice"

    Article Title: Loss of Caveolin 1 is Associated With the Expression of Aquaporin 1 and Bladder Dysfunction in Mice

    Journal: International Neurourology Journal

    doi: 10.5213/inj.2015.19.1.34

    Immunoblotting of aquaporin 1 (AQP1) and caveolin 1 (CAV1) in the wild type and CAV1 knockout (KO) mice urinary bladder. The anti-AQP antibodies recognize the 27- to 29-kDa bands that correspond to glycosylated AQPs. The anti-CAV1 antibodies recognize the 22-kDa band. Anti-GAPDH antibody recognizes the 42-kDa band. The expression of AQP1 was significantly increased in the CAV1 KO mice. The lower panel denotes the means±standard deviation of the 10 experiments for each condition, as determined by the densitometry relative to GAPDH. WT, wild type; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. * P
    Figure Legend Snippet: Immunoblotting of aquaporin 1 (AQP1) and caveolin 1 (CAV1) in the wild type and CAV1 knockout (KO) mice urinary bladder. The anti-AQP antibodies recognize the 27- to 29-kDa bands that correspond to glycosylated AQPs. The anti-CAV1 antibodies recognize the 22-kDa band. Anti-GAPDH antibody recognizes the 42-kDa band. The expression of AQP1 was significantly increased in the CAV1 KO mice. The lower panel denotes the means±standard deviation of the 10 experiments for each condition, as determined by the densitometry relative to GAPDH. WT, wild type; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. * P

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Standard Deviation

    38) Product Images from "Early growth response protein-1 mediates lipotoxicity-associated placental inflammation: role in maternal obesity"

    Article Title: Early growth response protein-1 mediates lipotoxicity-associated placental inflammation: role in maternal obesity

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    doi: 10.1152/ajpendo.00076.2013

    Protein expression of MAPKs and EGR-1 in term human placenta from lean and obese subjects. A and B : immunoblots ( A ) and densitometric quantitation ( B ) for total and phosphorylated p38, JNK1/2, and ERK1/2 in lean and obese placenta collected at term ( n = 11). Data are normalized to α-tubulin levels and expressed relative to the lean group (means ± SE). C : immunoblot analysis and densitometric quantitation of EGR-1 protein levels in lean or obese placenta ( n = 11). Densitometry values were normalized to GAPDH and expressed as means ± SE. *Significance, P ≤ 0.05.
    Figure Legend Snippet: Protein expression of MAPKs and EGR-1 in term human placenta from lean and obese subjects. A and B : immunoblots ( A ) and densitometric quantitation ( B ) for total and phosphorylated p38, JNK1/2, and ERK1/2 in lean and obese placenta collected at term ( n = 11). Data are normalized to α-tubulin levels and expressed relative to the lean group (means ± SE). C : immunoblot analysis and densitometric quantitation of EGR-1 protein levels in lean or obese placenta ( n = 11). Densitometry values were normalized to GAPDH and expressed as means ± SE. *Significance, P ≤ 0.05.

    Techniques Used: Expressing, Western Blot, Quantitation Assay

    39) Product Images from "Molecular and Phenotypic Characterisation of Paediatric Glioma Cell Lines as Models for Preclinical Drug Development"

    Article Title: Molecular and Phenotypic Characterisation of Paediatric Glioma Cell Lines as Models for Preclinical Drug Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005209

    Constitutive activation of key signalling pathways in glioma cell lines. Western blots analysis of c-Raf, phospho/total Erk1/2, phospho/total Akt, phospho/total GSK3β, phospho/total S6 and GAPDH as loading control in adult (LN229, A172, U118MG, U138MG, U87MG, SF268) and paediatric (SF188, KNS42, UW479, Res259, Res186) glioma cell lines.
    Figure Legend Snippet: Constitutive activation of key signalling pathways in glioma cell lines. Western blots analysis of c-Raf, phospho/total Erk1/2, phospho/total Akt, phospho/total GSK3β, phospho/total S6 and GAPDH as loading control in adult (LN229, A172, U118MG, U138MG, U87MG, SF268) and paediatric (SF188, KNS42, UW479, Res259, Res186) glioma cell lines.

    Techniques Used: Activation Assay, Western Blot

    40) Product Images from "Sensory Dysfunction of Bladder Mucosa and Bladder Oversensitivity in a Rat Model of Metabolic Syndrome"

    Article Title: Sensory Dysfunction of Bladder Mucosa and Bladder Oversensitivity in a Rat Model of Metabolic Syndrome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045578

    Alterations of receptor protein expression in the mucosa layer or smooth muscle layer of the bladder in controls and FFRs. Western blot analysis with specific antibodies to the TRPV1 receptor, purinergic P2X 2 receptor, and P2X 3 receptor of the rat mucosal layer and the purinergic P2X 1 receptor of the rat smooth muscle layer in controls and FFRs with different in cystometric presentations. A. TRPV1 receptor: The TRPV1 antibody produced a clear single band at 95kDa. B. Purinergic P2X 2 receptor C. Purinergic P2X 3 mature receptor: the predominant P2X 3 form (65kDa). D. Up-regulation of P2X3 native and intermediate polypeptides (up to 55kD) were shown in both FFR groups. E. Purinergic P2X 1 receptor Experiments were repeated two times and representative blots are shown. Data of proteins expression (ratios of signal intensities of investigated receptors relative to β-actin or GAPDH) were calculated with 8 samples in each group. These data of Mean ± SE were standardized and expressed in percentage in which the value of the control group is treated as 100%. Theses values were shown in the bar graph. An asterisk indicates a significant difference between controls and FFR groups (One-way ANOVA with Dunnett’s test, p
    Figure Legend Snippet: Alterations of receptor protein expression in the mucosa layer or smooth muscle layer of the bladder in controls and FFRs. Western blot analysis with specific antibodies to the TRPV1 receptor, purinergic P2X 2 receptor, and P2X 3 receptor of the rat mucosal layer and the purinergic P2X 1 receptor of the rat smooth muscle layer in controls and FFRs with different in cystometric presentations. A. TRPV1 receptor: The TRPV1 antibody produced a clear single band at 95kDa. B. Purinergic P2X 2 receptor C. Purinergic P2X 3 mature receptor: the predominant P2X 3 form (65kDa). D. Up-regulation of P2X3 native and intermediate polypeptides (up to 55kD) were shown in both FFR groups. E. Purinergic P2X 1 receptor Experiments were repeated two times and representative blots are shown. Data of proteins expression (ratios of signal intensities of investigated receptors relative to β-actin or GAPDH) were calculated with 8 samples in each group. These data of Mean ± SE were standardized and expressed in percentage in which the value of the control group is treated as 100%. Theses values were shown in the bar graph. An asterisk indicates a significant difference between controls and FFR groups (One-way ANOVA with Dunnett’s test, p

    Techniques Used: Expressing, Western Blot, Produced

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    shRNA:

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    Negative Control:

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    Article Snippet: .. MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma) or a control scrambled CshRNA (Sigma) was inserted into the pLKO-tet-puro vector (Addgene, Plasmid #21915). .. The viral vectors were produced in HEK293T cells as previously described ( , ).

    Article Title: FoxO3 increases miR-34a to cause palmitate-induced cholangiocyte lipoapoptosis
    Article Snippet: .. Lentiviral shRNA transductionshRNA silencing lentiviral pLKO.1-puro vector targeting FoxO3 and control GFP were obtained from Sigma and stable transfections were carried out as described ( ). .. FoxO3 shRNA #1 and #2 target the nucleotide sequences 2185-2205 and 1626-1648 of FoxO3 mRNA (NM_001455.1), respectively.

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  • 99
    Millipore gapdh proteins
    NF45 regulates survivin and cyclin E expression downstream of XIAP and <t>cIAP1.</t> (A) Immunofluorescence images showing the multinucleation phenotype of the d5 HeLa cell line (arrow, bottom left panel) compared to the c HeLa cell line. The same phenotype can be reproduced by the transient siRNA knockdown of NF45 for 72 h (arrow, bottom right panel and inset). Cells were stained with Alexa Fluor 568 phalloidin for F-actin and with Hoechst stain for nuclei. (B) c and d5 cell phenotypes were quantified by propidium iodide staining and flow cytometry analysis. The number of multinucleated cells expressed as a percentage of the total number of viable cells was quantified and normalized to that of the control cell line (bottom). The number of cells in G 2 /M phase is also shown. (C) Western blot and densitometry analyses showing increased survivin expression in HeLa cells treated with 50 nM NF45 siRNA for 96 h compared to control siRNA. (D) survivin expression was blunted by XIAP overexpression in NF45 knocked-down HeLa cells. The HeLa cells were transfected with 50 nM NF45 or control siRNA and transfected 48 h later with GFP-XIAP for an additional 48 h. The protein extracts were analyzed by Western blotting and densitometry for survivin, XIAP, NF45, and <t>GAPDH.</t> (E) NF45 regulates cyclin E expression. HeLa cells were treated with 50 nM NF45 or control siRNA for 96 h, and expression of the indicated proteins was analyzed by Western blotting and densitometry. (F) NF45 controls cyclin E protein levels through its regulation of cIAP1 IRES-mediated translation. The HeLa cells were treated with 100 nM Smac mimetic (SM) or DMSO for 24 h and transfected with a pcDNA3-GFP or pcDNA3-GFP-NF45R plasmid for an additional 24 h. Protein extracts were analyzed by Western blotting for NF45, cIAP1, cyclin E, and GAPDH expression, and densitometry was performed.
    Gapdh Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lc3b ii gapdh
    Autophagy caused by starvation does not require MAPK8/9 in immortalized MEFs. (a) RPS6KB1, p-Thr389 RPS6KB1, and TUBA expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation with EBSS containing 5mM glucose (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses <t>GFP-LC3B.</t> Puncta formation following incubation with EBSS containing 5 mM glucose (2 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and <t>GAPDH</t> in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the average of WT control condition (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p
    Lc3b Ii Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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.
    Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NF45 regulates survivin and cyclin E expression downstream of XIAP and cIAP1. (A) Immunofluorescence images showing the multinucleation phenotype of the d5 HeLa cell line (arrow, bottom left panel) compared to the c HeLa cell line. The same phenotype can be reproduced by the transient siRNA knockdown of NF45 for 72 h (arrow, bottom right panel and inset). Cells were stained with Alexa Fluor 568 phalloidin for F-actin and with Hoechst stain for nuclei. (B) c and d5 cell phenotypes were quantified by propidium iodide staining and flow cytometry analysis. The number of multinucleated cells expressed as a percentage of the total number of viable cells was quantified and normalized to that of the control cell line (bottom). The number of cells in G 2 /M phase is also shown. (C) Western blot and densitometry analyses showing increased survivin expression in HeLa cells treated with 50 nM NF45 siRNA for 96 h compared to control siRNA. (D) survivin expression was blunted by XIAP overexpression in NF45 knocked-down HeLa cells. The HeLa cells were transfected with 50 nM NF45 or control siRNA and transfected 48 h later with GFP-XIAP for an additional 48 h. The protein extracts were analyzed by Western blotting and densitometry for survivin, XIAP, NF45, and GAPDH. (E) NF45 regulates cyclin E expression. HeLa cells were treated with 50 nM NF45 or control siRNA for 96 h, and expression of the indicated proteins was analyzed by Western blotting and densitometry. (F) NF45 controls cyclin E protein levels through its regulation of cIAP1 IRES-mediated translation. The HeLa cells were treated with 100 nM Smac mimetic (SM) or DMSO for 24 h and transfected with a pcDNA3-GFP or pcDNA3-GFP-NF45R plasmid for an additional 24 h. Protein extracts were analyzed by Western blotting for NF45, cIAP1, cyclin E, and GAPDH expression, and densitometry was performed.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleotide Composition of Cellular Internal Ribosome Entry Sites Defines Dependence on NF45 and Predicts a Posttranscriptional Mitotic Regulon

    doi: 10.1128/MCB.00546-12

    Figure Lengend Snippet: NF45 regulates survivin and cyclin E expression downstream of XIAP and cIAP1. (A) Immunofluorescence images showing the multinucleation phenotype of the d5 HeLa cell line (arrow, bottom left panel) compared to the c HeLa cell line. The same phenotype can be reproduced by the transient siRNA knockdown of NF45 for 72 h (arrow, bottom right panel and inset). Cells were stained with Alexa Fluor 568 phalloidin for F-actin and with Hoechst stain for nuclei. (B) c and d5 cell phenotypes were quantified by propidium iodide staining and flow cytometry analysis. The number of multinucleated cells expressed as a percentage of the total number of viable cells was quantified and normalized to that of the control cell line (bottom). The number of cells in G 2 /M phase is also shown. (C) Western blot and densitometry analyses showing increased survivin expression in HeLa cells treated with 50 nM NF45 siRNA for 96 h compared to control siRNA. (D) survivin expression was blunted by XIAP overexpression in NF45 knocked-down HeLa cells. The HeLa cells were transfected with 50 nM NF45 or control siRNA and transfected 48 h later with GFP-XIAP for an additional 48 h. The protein extracts were analyzed by Western blotting and densitometry for survivin, XIAP, NF45, and GAPDH. (E) NF45 regulates cyclin E expression. HeLa cells were treated with 50 nM NF45 or control siRNA for 96 h, and expression of the indicated proteins was analyzed by Western blotting and densitometry. (F) NF45 controls cyclin E protein levels through its regulation of cIAP1 IRES-mediated translation. The HeLa cells were treated with 100 nM Smac mimetic (SM) or DMSO for 24 h and transfected with a pcDNA3-GFP or pcDNA3-GFP-NF45R plasmid for an additional 24 h. Protein extracts were analyzed by Western blotting for NF45, cIAP1, cyclin E, and GAPDH expression, and densitometry was performed.

    Article Snippet: Coimmunoprecipitations of cIAP1, XIAP, and GAPDH proteins were performed at 4°C overnight using protein G/protein A-agarose beads (EMD Chemicals) coated with, respectively, the antibodies anti-cIAP1 (R & D Systems, MN) at a 1:150 dilution, anti-GST-XIAP ( ) (Aegera) at a 1:150 dilution, and anti-GAPDH (clone 6C5; Advanced ImmunoChemical, CA) at a 1:250 dilution.

    Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Western Blot, Over Expression, Transfection, Plasmid Preparation

    NF45 regulates XIAP translation through interaction with its IRES. (A) Western blots of endogenous XIAP and cIAP1 protein expression in d5 cells relative to that in c cells. The blots and densitometry analyses are representative of at least three experiments. (B) NF45 reexpression in NF45-deficient HeLa cells rescues XIAP protein expression. The HeLa cells were transfected with 50 nM control nontargeting siRNA or NF45 siRNA (d5 siRNA) for 48 h, followed by the overexpression of GFP or GFP-NF45R for an additional 48 h. The cell extracts were analyzed by Western blotting for NF45, XIAP, cIAP1, and GAPDH expression, and protein expression was quantified. (C) De novo protein expression of cIAP1 and XIAP in NF45-deficient cells. c cells were transfected with 50 nM control or NF45 siRNA and were pulse-labeled with [ 35 S]methionine. 35 S-labeled and Coomassie blue-stained total protein, as well as specific cIAP1, XIAP, and GAPDH immunoprecipitates, are shown. (D) NF45 interacts specifically with the XIAP IRES. NF45 and PABP Western blotting of an RNA affinity chromatography preparation was performed using the XIAP IRES or hemoglobin RNA.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleotide Composition of Cellular Internal Ribosome Entry Sites Defines Dependence on NF45 and Predicts a Posttranscriptional Mitotic Regulon

    doi: 10.1128/MCB.00546-12

    Figure Lengend Snippet: NF45 regulates XIAP translation through interaction with its IRES. (A) Western blots of endogenous XIAP and cIAP1 protein expression in d5 cells relative to that in c cells. The blots and densitometry analyses are representative of at least three experiments. (B) NF45 reexpression in NF45-deficient HeLa cells rescues XIAP protein expression. The HeLa cells were transfected with 50 nM control nontargeting siRNA or NF45 siRNA (d5 siRNA) for 48 h, followed by the overexpression of GFP or GFP-NF45R for an additional 48 h. The cell extracts were analyzed by Western blotting for NF45, XIAP, cIAP1, and GAPDH expression, and protein expression was quantified. (C) De novo protein expression of cIAP1 and XIAP in NF45-deficient cells. c cells were transfected with 50 nM control or NF45 siRNA and were pulse-labeled with [ 35 S]methionine. 35 S-labeled and Coomassie blue-stained total protein, as well as specific cIAP1, XIAP, and GAPDH immunoprecipitates, are shown. (D) NF45 interacts specifically with the XIAP IRES. NF45 and PABP Western blotting of an RNA affinity chromatography preparation was performed using the XIAP IRES or hemoglobin RNA.

    Article Snippet: Coimmunoprecipitations of cIAP1, XIAP, and GAPDH proteins were performed at 4°C overnight using protein G/protein A-agarose beads (EMD Chemicals) coated with, respectively, the antibodies anti-cIAP1 (R & D Systems, MN) at a 1:150 dilution, anti-GST-XIAP ( ) (Aegera) at a 1:150 dilution, and anti-GAPDH (clone 6C5; Advanced ImmunoChemical, CA) at a 1:250 dilution.

    Techniques: Western Blot, Expressing, Transfection, Over Expression, Labeling, Staining, Affinity Chromatography

    Autophagy caused by starvation does not require MAPK8/9 in immortalized MEFs. (a) RPS6KB1, p-Thr389 RPS6KB1, and TUBA expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation with EBSS containing 5mM glucose (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the average of WT control condition (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Autophagy caused by starvation does not require MAPK8/9 in immortalized MEFs. (a) RPS6KB1, p-Thr389 RPS6KB1, and TUBA expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation with EBSS containing 5mM glucose (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the average of WT control condition (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Expressing, Incubation, Transduction, Plasmid Preparation, Fluorescence, Microscopy

    Autophagy caused by MTOR inhibition does not require MAPK8/9 in immortalized MEFs. (a) The amount of RPS6KB1, p-Thr389 RPS6KB1, and TUBA (tubulin alpha) in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation without or with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation of the cells with 250 nM torin 1 (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) without or with 250 nM torin 1 in the absence or presence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control condition (first lane). The ‘Change in MAP1LC3B-II’ was calculated by subtracting MAP1LC3B-II:GAPDH (media+ CQ condition) from MAP1LC3B-II:GAPDH (torin 1+ CQ condition). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Autophagy caused by MTOR inhibition does not require MAPK8/9 in immortalized MEFs. (a) The amount of RPS6KB1, p-Thr389 RPS6KB1, and TUBA (tubulin alpha) in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation without or with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation of the cells with 250 nM torin 1 (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) without or with 250 nM torin 1 in the absence or presence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control condition (first lane). The ‘Change in MAP1LC3B-II’ was calculated by subtracting MAP1LC3B-II:GAPDH (media+ CQ condition) from MAP1LC3B-II:GAPDH (torin 1+ CQ condition). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Inhibition, Incubation, Transduction, Plasmid Preparation, Fluorescence, Microscopy, Expressing

    Autophagy caused by starvation does not require MAPK8/9 in primary MEFs. (a) (Z)-4-Hydroxytamoxifen-treated primary Rosa- Cre ERT (WT) MEFs and Rosa- Cre ERT Mapk8 LoxP/LoxP mapk9 −/- MEFs were examined by immunoblot analysis by probing with antibodies to MAPK8/9 and TUBA. (b) WT and mapk8 −/- mapk9 −/- primary MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 25 µm. (c) LC3B and GAPDH expression by WT and mapk8 −/- mapk9 −/- primary MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Autophagy caused by starvation does not require MAPK8/9 in primary MEFs. (a) (Z)-4-Hydroxytamoxifen-treated primary Rosa- Cre ERT (WT) MEFs and Rosa- Cre ERT Mapk8 LoxP/LoxP mapk9 −/- MEFs were examined by immunoblot analysis by probing with antibodies to MAPK8/9 and TUBA. (b) WT and mapk8 −/- mapk9 −/- primary MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 25 µm. (c) LC3B and GAPDH expression by WT and mapk8 −/- mapk9 −/- primary MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Transduction, Plasmid Preparation, Incubation, Fluorescence, Microscopy, Expressing

    Effect of starvation and MTOR inhibition on MAPK8/9 activation is not sufficient to cause autophagy. (a and b) MAPK8/9 activation in immortalized MEFs was examined by immunoblot analysis of p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH in cells after incubation (2 or 4 h) with EBSS containing 5 mM glucose (a) or 250 nM torin 1 (b). Lanes 1 and 2 represent positive and negative controls: lysates of WT MEFs exposed to 60 J/m 2 UV and mapk8 −/- mapk9 −/- MEFs, respectively. (c) WT and mapk8 −/- mapk9 −/- immortalized MEFs were exposed to UV radiation (60 J/m 2 ) and cell extracts were prepared at 45 min post-irradiation. The expression of LC3B, p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH was examined by immunoblot analysis.

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Effect of starvation and MTOR inhibition on MAPK8/9 activation is not sufficient to cause autophagy. (a and b) MAPK8/9 activation in immortalized MEFs was examined by immunoblot analysis of p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH in cells after incubation (2 or 4 h) with EBSS containing 5 mM glucose (a) or 250 nM torin 1 (b). Lanes 1 and 2 represent positive and negative controls: lysates of WT MEFs exposed to 60 J/m 2 UV and mapk8 −/- mapk9 −/- MEFs, respectively. (c) WT and mapk8 −/- mapk9 −/- immortalized MEFs were exposed to UV radiation (60 J/m 2 ) and cell extracts were prepared at 45 min post-irradiation. The expression of LC3B, p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH was examined by immunoblot analysis.

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Inhibition, Activation Assay, Incubation, Irradiation, Expressing

    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