gapdh Search Results


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  • 99
    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
    Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15716 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc gapdh
    GluN2A and GluN2B protein levels in adult human temporal cortical tissue homogenate show an age-dependent decline. (A) Representative examples of GluN2A blot showing GluN2A and GluN2A-S bands. Quantification for GluN2A (B) and GluN2A-S (C), and GluN2B (B, from blots shown in Fig. 1E ) relative to the constitutive protein <t>GAPDH.</t> Spearman’s coefficient of correlation (r) and p-value (p) are indicated for each measure. (E) Representative blots of GluN2A and GluN2B subunits co-immunoprecipitated with <t>PSD-95</t> using human temporal cortical tissue lysates. Symbols in B, C and D correspond to the neurological condition necessitating neurosurgery as depicted in Figure 1 .
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 30846 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Santa Cruz Biotechnology gapdh
    LGP2 enhancement of <t>MDA5-mediated</t> signaling requires ATP hydrolysis, RNA binding, and intact MDA5 oligomerization A. LGP2 ATP hydrolysis and RNA binding are required for MDA5 co-activation. HEK293T cells were transfected with −110 IFNβ-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for MDA5, LGP2, or LGP2 mutants (MIIa and MIII). MDA5 was transfected at a constant 25ng plasmid/well, while the amount of LGP2 was titrated. After 24hr, cells were transfected with 5µg/ml poly(I:C) for 6hr prior to harvesting. In each plot, the activity of MDA5 stimulated by poly(I:C) is normalized to 100%. Expression levels of MDA5, LGP2, and <t>GAPDH</t> were analyzed by immunoblotting with specific antisera. B. Signal transduction by MDA5 monomer interface mutants. IFNβ luciferase assays similar to A., but cells were transfected with 25ng of MDA5-WT or indicated MDA5 monomer-interface mutants: M570R/D572R, I841R/E842R, and M570R/D572R/I841R/E842R. C. LGP2 does not augment signaling by MDA5 monomer interface mutants. IFNβ luciferase assay similar to A, but cells were transfected with 25ng of MDA5-WT or MDA5 monomer-interface mutants: 570/572, 841/842, and 570/572/841/842, and a single enhancing concentration of LGP2 (4ng). D. LGP2 does not augment signaling by MDA5-570/572 mutant. IFNβ luciferase assay (as in A,B,C). HEK293T cells were transfected with 25ng of MDA5-WT or MDA5-570/572. The amount of LGP2 transfected was titrated at 0.16, 0.8, 4, 20, 100, and 500ng. No concentration of LGP2 was able to enhance MDA5-570572 mediated signaling.
    Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 31521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gapdh  (Abcam)
    95
    Abcam gapdh
    Lentivirus-mediated PLCγ1 shRNA could suppress migration in human gastric adenocarcinoma BGC-823 cells Cells were transduced with lentivirus-mediated PLCγ1 shRNA2/3 vectors. ( A ) The formation of membrane ruffles was detected using Ruffling assay as described in Materials and Methods. The cell nuclei were stained DAPI (blue) and the membrane ruffles were stained rhodamine-conjugated phalloidin (red). Scale bar = 10 μm. ( B and C ) The migration ability was measured using Transwell assay (B, magnification × 100) and Scratch assay (C, magnification × 400) as described in Materials and Methods. ( D ) The protein levels of MMP2, <t>MMP9,</t> E-cadherin, N-cadherin, snail, slug, and <t>GAPDH</t> were detected with Western blotting analysis, and the pro and active forms of MMP2/9 were observed using gelatin zymography assay as described in Materials and Methods. ( E ) The mRNA levels of PLCG1, MMP2, MMP9, CDHI, CDH2, SNAIL, SLUG, and GAPDH were detected using Real-time PCR analysis as described in Materials and Methods. ( F ) The level of VEGF in extracellular matrix was detected using ELISA as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (* P
    Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 18727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Santa Cruz Biotechnology anti gapdh
    Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of <t>Fgf21,</t> Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to <t>Gapdh</t> and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P
    Anti Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 11900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gene exp gapdh mm99999915 g1
    Regulation of the expression of metastasis-related genes by Akt isoforms. GEO cells expressing non-targeting shRNA (sh) or <t>Akt1,</t> Akt2 or Akt3 shRNA were treated with Dox for 5 days and cDNA samples were subjected to real-time <t>PCR</t> using the Human Tumor Metastasis RT 2 Profiler PCR array from Super Array Biosciences (Qiagen). Top panel: Venn diagram showing differentially expressed metastatic genes on knockdown of each of the three Akt isoforms. Genes shown in red have higher expression in Akt isoform knockdown cells relative to non-targeting shRNA (sh) expressing cells, whereas genes in green show reduced expression. Lower panel: list of genes that showed differential expression ( > two-fold) on loss of individual Akt isoforms. MTSS1 is shown in bold.
    Gene Exp Gapdh Mm99999915 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8768 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti gapdh
    HGF stimulation is abrogated by <t>IQSEC1</t> depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and <t>GAPDH</t> (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.
    Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 9865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gene exp gapdh hs99999905 m1
    Quantitative real time polymerase chain reaction <t>(qRT-PCR)</t> of patient <t>survivin</t> levels. A: qRT-PCR was performed to compare survivin expression between tumor and adjacent squamous epithelial tissue. B: On average, tumor samples showed 3× greater survivin expression than paired adjacent tissue.
    Gene Exp Gapdh Hs99999905 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher gapdh
    Effect of G17 on Snail expression in gastric cancer cells . (A) AGSE cells were treated as in 1A or (B) 1B above and subjected to Western Blot analysis utilizing antibodies against Snail and <t>GAPDH</t> (as control). (C) Western Blot analysis of cell extracts with the indicated antibodies, treated with 100 nM G17 for 1 hour, following an overnight pretreatment with 100 nM YM 022. (D) Subconfluent AGSE cells were transiently transfected with Snail-luciferase vector (Snail-luc) along with <t>β-Gal</t> vector (for normalization of transfection). Forty-eight hours after transfection, cells were treated overnight in the presence (+) or absence (-) of 100 nM G17, and luciferase and β-Gal assays were performed. The RLU/β-Gal values were represented as percent control, considering the untreated samples as 100%. Each transfection was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments.
    Gapdh, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 14534 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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 9150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gene exp gapdh hs02758991 g1
    Expression of <t>STAT5b</t> mRNA and protein in pancreatic cancer cells. (A) Quantitative <t>RT-PCR</t> analysis of STAT5b mRNA levels. STAT5b mRNA expressed in all pancreatic cancer cell lines, and the levels were highest in PANC-1 cells, second highest in Capan-1 cells, and lowest in PK-45H cells. In PANC-1 cells, STAT5b mRNA levels were ~13.5-fold higher than in PK-45H cells. (B) Western blot analysis of STAT5b protein levels. STAT5b protein was detected in all cell lines consistent with the RT-PCR results and the levels were highest in PANC-1 cells and second highest in Capan-1 cells.
    Gene Exp Gapdh Hs02758991 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mouse monoclonal antibody
    Chromatin architecture of the Reg locus in adult cortex and pancreas. ( A ) Levels of cohesin were assayed by immunoblot with <t>SA1</t> and Rad21 antibodies in pancreas from wild-type and SA1 heterozygous mice (two individuals per genotype). <t>GAPDH</t> serves as loading control. ( B ) Table showing the four genes differentially expressed (FDR
    Mouse Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc rabbit anti gapdh
    K nockdown of <t>FTO</t> does not significantly change mRNA levels of genes involved in miRNA biogenesis. The steady-state mRNA levels of DICER , DROSHA , DGCR8 and ADAR were analyzed by qRT-PCR in cells treated with scrambled (scr) and FTO- specific siRNAs, respectively. <t>GAPDH</t> was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. FTO kd, FTO -specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.
    Rabbit Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc glyceraldehyde 3 phosphate dehydrogenase gapdh
    P2X7 and EGF Receptor Stimulation Regulates the Expression of DUSP6 in Cerebellar Granule Neurons and Astrocytes ) for different times and DUSP6 and <t>Glyceraldehyde-3-phosphate</t> dehydrogenase <t>(GAPDH)</t> were detected in the total lysates by immunoblotting. (A) Immunoblots of a representative time-course experiment for each effector. (B) Cells were incubated with or without the effectors, 300 μM BzATP or 100 ng/mL EGF, for 30 min, fixed and the presence of DUSP6 was detected by immunocytochemistry. Representative immunofluorescence images are shown. Scale bars represent 50 μm. (C) The diagrams represent the quantification of the time courses obtained by normalization to the corresponding GAPDH levels. The data are represented as the means ± SEM from three independent experiments performed on different cultures. *** p
    Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 2135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology glyceraldehyde 3 phosphate dehydrogenase gapdh
    Atorvastatin effect on the expression of smooth muscle actin. <t>GAPDH:</t> <t>Glyceraldehyde-3-phosphate</t> dehydrogenase; SMA: Smooth muscle actin.
    Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0207190

    Figure Lengend 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

    Article Snippet: Protein analysis through antibody-microarray CRMP-5-, PKB- and GAPDH- (as a loading control) Abs (GAPDH-AB, Sigma Aldrich, USA) (0.1 mg/mL) were spotted on a glass-nitrocellulose 16 multi-pad slide (Oncyte, Grace Bio-Labs, USA) with nine technical replicates per subarray using a non-contact array spotter (ciFLEXARRAYER 3, Scienion, Germany).

    Techniques: Microarray

    GluN2A and GluN2B protein levels in adult human temporal cortical tissue homogenate show an age-dependent decline. (A) Representative examples of GluN2A blot showing GluN2A and GluN2A-S bands. Quantification for GluN2A (B) and GluN2A-S (C), and GluN2B (B, from blots shown in Fig. 1E ) relative to the constitutive protein GAPDH. Spearman’s coefficient of correlation (r) and p-value (p) are indicated for each measure. (E) Representative blots of GluN2A and GluN2B subunits co-immunoprecipitated with PSD-95 using human temporal cortical tissue lysates. Symbols in B, C and D correspond to the neurological condition necessitating neurosurgery as depicted in Figure 1 .

    Journal: bioRxiv

    Article Title: Age dependent changes in synaptic NMDA receptor composition in adult human cortical neurons

    doi: 10.1101/2020.01.21.913475

    Figure Lengend Snippet: GluN2A and GluN2B protein levels in adult human temporal cortical tissue homogenate show an age-dependent decline. (A) Representative examples of GluN2A blot showing GluN2A and GluN2A-S bands. Quantification for GluN2A (B) and GluN2A-S (C), and GluN2B (B, from blots shown in Fig. 1E ) relative to the constitutive protein GAPDH. Spearman’s coefficient of correlation (r) and p-value (p) are indicated for each measure. (E) Representative blots of GluN2A and GluN2B subunits co-immunoprecipitated with PSD-95 using human temporal cortical tissue lysates. Symbols in B, C and D correspond to the neurological condition necessitating neurosurgery as depicted in Figure 1 .

    Article Snippet: Membranes were blocked in 5% (w/v) non-fat milk for 1hr at room temperature and incubated overnight at 4°C in 5% (w/v) bovine serum albumin (BSA) containing 0.1% (v/v) Tween-20 and one of the following primary antibodies: anti-NMDAR2A (ab133265; 1:1000; Abcam); anti-NMDAR2B (610416; 1:1000; BD Neurosciences), anti-PSD-95 (D27E11; 1:1000; CST), and GAPDH (D16H11; 1:1000; CST).

    Techniques: Immunoprecipitation

    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Journal: Neurochemistry International

    Article Title: Effect of Glutamate and Riluzole on Manganese-Induced Apoptotic Cell Signaling in Neuronally Differentiated Mouse P19 Cells

    doi: 10.1016/j.neuint.2012.04.015

    Figure Lengend Snippet: Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Article Snippet: Phospho-JNK antibody, anti-GAPDH antibody and secondary antibody for Western blots were obtained from Cell Signaling (Danvers, MA) and Western lightning plus substrate to develop immunoblots was from Perkin Elmer (Waltham, MA).

    Techniques: Western Blot

    The miR-320d and FoxM1 expression level in GCA tissues and the adjacent normal tissues. RT-qPCR analysis of miR-320d expression level ( a ) and FoxM1 mRNA level ( b ) in 60 GCA tissues and the adjacent non-cancerous tissues. c Correlation between miR-320d expression and FoxM1 mRNA expression in 60 GCA tissues ( r = − 2.94, P = 0.023). Representative IHC images ( d ) and western-blot analysis ( e ) of FoxM1 protein in GCA and normal tissues. f IHC scores of FoxM1 protein in GCA and normal tissues evaluated by the independent pathologists (n = 4). g Quantification of FoxM1 level by western-blot (n = 6). GAPDH was normalized as 100%. The * represents significant difference from GCA tissues to the normal tissues (**: P

    Journal: Cell & Bioscience

    Article Title: MicroRNA-320d regulates tumor growth and invasion by promoting FoxM1 and predicts poor outcome in gastric cardiac adenocarcinoma

    doi: 10.1186/s13578-020-00439-7

    Figure Lengend Snippet: The miR-320d and FoxM1 expression level in GCA tissues and the adjacent normal tissues. RT-qPCR analysis of miR-320d expression level ( a ) and FoxM1 mRNA level ( b ) in 60 GCA tissues and the adjacent non-cancerous tissues. c Correlation between miR-320d expression and FoxM1 mRNA expression in 60 GCA tissues ( r = − 2.94, P = 0.023). Representative IHC images ( d ) and western-blot analysis ( e ) of FoxM1 protein in GCA and normal tissues. f IHC scores of FoxM1 protein in GCA and normal tissues evaluated by the independent pathologists (n = 4). g Quantification of FoxM1 level by western-blot (n = 6). GAPDH was normalized as 100%. The * represents significant difference from GCA tissues to the normal tissues (**: P

    Article Snippet: The total proteins were incubated with FoxM1 primary antibody (1:1000, Abcam, USA) and GAPDH antibody (Cell signaling, USA) at 4 °C for overnight.

    Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Western Blot

    The regulatory relationship of miR-320d and FoxM1 in GCA cell lines. a RT-qPCR analysis of miR-320d and FoxM1 expression levels in GCA cell SK-GT2 and OE-19. b The expression of miR-320d in OE-19 cell that transfected with miR-320d mimics or in SK-GT2 cell with miR-320d inhibitor transfection. Western blot images ( c ) or quantification ( d ) of FoxM1 expression in GCA cells with or without transfection. The empty vector (EV) was set as control, and GAPDH was normalized as 100%. The * represents significant difference from miR-320d vector-transfected cells to EV-transfected cells (*: P

    Journal: Cell & Bioscience

    Article Title: MicroRNA-320d regulates tumor growth and invasion by promoting FoxM1 and predicts poor outcome in gastric cardiac adenocarcinoma

    doi: 10.1186/s13578-020-00439-7

    Figure Lengend Snippet: The regulatory relationship of miR-320d and FoxM1 in GCA cell lines. a RT-qPCR analysis of miR-320d and FoxM1 expression levels in GCA cell SK-GT2 and OE-19. b The expression of miR-320d in OE-19 cell that transfected with miR-320d mimics or in SK-GT2 cell with miR-320d inhibitor transfection. Western blot images ( c ) or quantification ( d ) of FoxM1 expression in GCA cells with or without transfection. The empty vector (EV) was set as control, and GAPDH was normalized as 100%. The * represents significant difference from miR-320d vector-transfected cells to EV-transfected cells (*: P

    Article Snippet: The total proteins were incubated with FoxM1 primary antibody (1:1000, Abcam, USA) and GAPDH antibody (Cell signaling, USA) at 4 °C for overnight.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Plasmid Preparation

    LGP2 enhancement of MDA5-mediated signaling requires ATP hydrolysis, RNA binding, and intact MDA5 oligomerization A. LGP2 ATP hydrolysis and RNA binding are required for MDA5 co-activation. HEK293T cells were transfected with −110 IFNβ-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for MDA5, LGP2, or LGP2 mutants (MIIa and MIII). MDA5 was transfected at a constant 25ng plasmid/well, while the amount of LGP2 was titrated. After 24hr, cells were transfected with 5µg/ml poly(I:C) for 6hr prior to harvesting. In each plot, the activity of MDA5 stimulated by poly(I:C) is normalized to 100%. Expression levels of MDA5, LGP2, and GAPDH were analyzed by immunoblotting with specific antisera. B. Signal transduction by MDA5 monomer interface mutants. IFNβ luciferase assays similar to A., but cells were transfected with 25ng of MDA5-WT or indicated MDA5 monomer-interface mutants: M570R/D572R, I841R/E842R, and M570R/D572R/I841R/E842R. C. LGP2 does not augment signaling by MDA5 monomer interface mutants. IFNβ luciferase assay similar to A, but cells were transfected with 25ng of MDA5-WT or MDA5 monomer-interface mutants: 570/572, 841/842, and 570/572/841/842, and a single enhancing concentration of LGP2 (4ng). D. LGP2 does not augment signaling by MDA5-570/572 mutant. IFNβ luciferase assay (as in A,B,C). HEK293T cells were transfected with 25ng of MDA5-WT or MDA5-570/572. The amount of LGP2 transfected was titrated at 0.16, 0.8, 4, 20, 100, and 500ng. No concentration of LGP2 was able to enhance MDA5-570572 mediated signaling.

    Journal: Molecular cell

    Article Title: The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly

    doi: 10.1016/j.molcel.2014.07.003

    Figure Lengend Snippet: LGP2 enhancement of MDA5-mediated signaling requires ATP hydrolysis, RNA binding, and intact MDA5 oligomerization A. LGP2 ATP hydrolysis and RNA binding are required for MDA5 co-activation. HEK293T cells were transfected with −110 IFNβ-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for MDA5, LGP2, or LGP2 mutants (MIIa and MIII). MDA5 was transfected at a constant 25ng plasmid/well, while the amount of LGP2 was titrated. After 24hr, cells were transfected with 5µg/ml poly(I:C) for 6hr prior to harvesting. In each plot, the activity of MDA5 stimulated by poly(I:C) is normalized to 100%. Expression levels of MDA5, LGP2, and GAPDH were analyzed by immunoblotting with specific antisera. B. Signal transduction by MDA5 monomer interface mutants. IFNβ luciferase assays similar to A., but cells were transfected with 25ng of MDA5-WT or indicated MDA5 monomer-interface mutants: M570R/D572R, I841R/E842R, and M570R/D572R/I841R/E842R. C. LGP2 does not augment signaling by MDA5 monomer interface mutants. IFNβ luciferase assay similar to A, but cells were transfected with 25ng of MDA5-WT or MDA5 monomer-interface mutants: 570/572, 841/842, and 570/572/841/842, and a single enhancing concentration of LGP2 (4ng). D. LGP2 does not augment signaling by MDA5-570/572 mutant. IFNβ luciferase assay (as in A,B,C). HEK293T cells were transfected with 25ng of MDA5-WT or MDA5-570/572. The amount of LGP2 transfected was titrated at 0.16, 0.8, 4, 20, 100, and 500ng. No concentration of LGP2 was able to enhance MDA5-570572 mediated signaling.

    Article Snippet: Lysates were analyzed by immunoblot with a monoclonal antibody to MDA5 ( ) and with mouse anti-FLAGM2 to detect MDA5-C and LGP2 expression (Sigma), and with GAPDH as a loading control (Santa Cruz Biotechnologies).

    Techniques: RNA Binding Assay, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Transduction, Concentration Assay, Mutagenesis

    TRF2 binding on hTERT promoter is independent of telomere looping A . Schematic for insertion of Gaussia luciferase gene, driven by (+33 to-1276) bp hTERT promoter inserted at CCR5 safe harbour locus (46Mb away from nearest telomere using CRISPR/Cas9) in HEK293T cells. B . Effect of TRF2 silencing or over-expression of DNA binding mutants of TRF2, TRF2-DelB, TRF2-DelM and TRF2-DelB DelM on Gaussia luciferase activity, normalized over total protein C . qRT-PCR following TRF2 ChIP on the exogenously inserted hTERT promoter at CCR5 locus ; normalized over mock (IgG) ( GAPDH promoter-negative control for TRF2 occupancy) D . qRT-PCR following ChIP for shelterin complex proteins TRF1, POT1 and RAP1, on hTERT promoter insert in HEK293T cells E . qRT-PCR following ChIP for TRF1, POT1 and RAP1 on endogenous hTERT promoter in HT1080 cells (in D-E: Chromosome 5p (interstitial telomeric sequence) ITS site-positive control and GAPDH promoter-negative control). All error bars represent ± standard deviations from mean values and p values have been calculated by paired /un paired T-test. (*: p

    Journal: bioRxiv

    Article Title: Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2

    doi: 10.1101/2020.01.15.907626

    Figure Lengend Snippet: TRF2 binding on hTERT promoter is independent of telomere looping A . Schematic for insertion of Gaussia luciferase gene, driven by (+33 to-1276) bp hTERT promoter inserted at CCR5 safe harbour locus (46Mb away from nearest telomere using CRISPR/Cas9) in HEK293T cells. B . Effect of TRF2 silencing or over-expression of DNA binding mutants of TRF2, TRF2-DelB, TRF2-DelM and TRF2-DelB DelM on Gaussia luciferase activity, normalized over total protein C . qRT-PCR following TRF2 ChIP on the exogenously inserted hTERT promoter at CCR5 locus ; normalized over mock (IgG) ( GAPDH promoter-negative control for TRF2 occupancy) D . qRT-PCR following ChIP for shelterin complex proteins TRF1, POT1 and RAP1, on hTERT promoter insert in HEK293T cells E . qRT-PCR following ChIP for TRF1, POT1 and RAP1 on endogenous hTERT promoter in HT1080 cells (in D-E: Chromosome 5p (interstitial telomeric sequence) ITS site-positive control and GAPDH promoter-negative control). All error bars represent ± standard deviations from mean values and p values have been calculated by paired /un paired T-test. (*: p

    Article Snippet: After blocking the membrane was incubated with primary antibodies-anti-TRF2 antibody (Novus Biological), anti-TERT antibody (Abcam), anti-REST(Millipore), anti-EZH2(CST) and anti-GAPDH antibody (Santa-cruz).

    Techniques: Binding Assay, Luciferase, CRISPR, Over Expression, Activity Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, Sequencing, Positive Control

    Effects of Meth on dopamine receptors and sigma-1 receptor in CD4 + T-cells. ( A ) CD4 + T-cells were untreated or treated with 100 µM Meth for different time points (5 mins-24 hours), lysed and the protein extracts were analyzed for the expression of various dopamine receptors and sigma-1 receptor. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( B ) Fold change in the pixel density of sigma-1 receptor expression in ( A ). All values normalized to untreated sample. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( C ) CD4 + T-cells were untreated or treated with 10 µM sigma-1 receptor inhibitor (σ1 R inh.) for 1 hour, then treated with or without 100 µM Meth for 1 hour, followed by lysis and analysis of protein extracts for the indicated activated signaling molecules by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( D ) HIV-1 p24 titer on day 3 after HIV-1 infection in unstimulated and stimulated CD4 + T-cells pretreated with or without sigma-1 receptor inhibitor (σ1 R inh.) and treated in the presence or absence of Meth. Data represent the mean ± SD of 3 independent experiments (**p ≤ 0.01, ***p ≤ 0.001).

    Journal: Scientific Reports

    Article Title: Methamphetamine functions as a novel CD4+ T-cell activator via the sigma-1 receptor to enhance HIV-1 infection

    doi: 10.1038/s41598-018-35757-x

    Figure Lengend Snippet: Effects of Meth on dopamine receptors and sigma-1 receptor in CD4 + T-cells. ( A ) CD4 + T-cells were untreated or treated with 100 µM Meth for different time points (5 mins-24 hours), lysed and the protein extracts were analyzed for the expression of various dopamine receptors and sigma-1 receptor. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( B ) Fold change in the pixel density of sigma-1 receptor expression in ( A ). All values normalized to untreated sample. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( C ) CD4 + T-cells were untreated or treated with 10 µM sigma-1 receptor inhibitor (σ1 R inh.) for 1 hour, then treated with or without 100 µM Meth for 1 hour, followed by lysis and analysis of protein extracts for the indicated activated signaling molecules by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( D ) HIV-1 p24 titer on day 3 after HIV-1 infection in unstimulated and stimulated CD4 + T-cells pretreated with or without sigma-1 receptor inhibitor (σ1 R inh.) and treated in the presence or absence of Meth. Data represent the mean ± SD of 3 independent experiments (**p ≤ 0.01, ***p ≤ 0.001).

    Article Snippet: GW182, D1DR, D2DR, D3DR, D4DR, Sigma-1 Receptor, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Expressing, Lysis, Western Blot, Infection

    Meth induced degradation of Ago1 and altered structural integrity of P-bodies: ( A ) CD4 + T-cells were untreated or treated with Meth (100 µM) for 0, 4 and 24 hours, lysed and Ago1 expression was analyzed by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S4 ( B ) CD4 + T-cell lysates in ( A ) were immunoprecipitated with Ago1 antibody and subjected to Western blot analysis using Ubiquitin antibody. Ago1 served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. ( C ) CD4 + T-cell lysates in (A) were immunoprecipitated with GW182 antibody (upper panel) or Ago1 antibody (lower panel) and subjected to Western blot analysis using Ago1 (upper panel) or GW182 (lower panel) antibodies. B-Actin served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S4 ( D ) Confocal images of GW182 and Ago1 interaction in CD4 + T-cells, untreated or treated with Meth (100 µM) for 24 hours. Scale bar = 10 µm. Results are representative of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Methamphetamine functions as a novel CD4+ T-cell activator via the sigma-1 receptor to enhance HIV-1 infection

    doi: 10.1038/s41598-018-35757-x

    Figure Lengend Snippet: Meth induced degradation of Ago1 and altered structural integrity of P-bodies: ( A ) CD4 + T-cells were untreated or treated with Meth (100 µM) for 0, 4 and 24 hours, lysed and Ago1 expression was analyzed by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S4 ( B ) CD4 + T-cell lysates in ( A ) were immunoprecipitated with Ago1 antibody and subjected to Western blot analysis using Ubiquitin antibody. Ago1 served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. ( C ) CD4 + T-cell lysates in (A) were immunoprecipitated with GW182 antibody (upper panel) or Ago1 antibody (lower panel) and subjected to Western blot analysis using Ago1 (upper panel) or GW182 (lower panel) antibodies. B-Actin served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S4 ( D ) Confocal images of GW182 and Ago1 interaction in CD4 + T-cells, untreated or treated with Meth (100 µM) for 24 hours. Scale bar = 10 µm. Results are representative of 3 independent experiments.

    Article Snippet: GW182, D1DR, D2DR, D3DR, D4DR, Sigma-1 Receptor, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Expressing, Western Blot, Immunoprecipitation

    Lentivirus-mediated PLCγ1 shRNA could suppress migration in human gastric adenocarcinoma BGC-823 cells Cells were transduced with lentivirus-mediated PLCγ1 shRNA2/3 vectors. ( A ) The formation of membrane ruffles was detected using Ruffling assay as described in Materials and Methods. The cell nuclei were stained DAPI (blue) and the membrane ruffles were stained rhodamine-conjugated phalloidin (red). Scale bar = 10 μm. ( B and C ) The migration ability was measured using Transwell assay (B, magnification × 100) and Scratch assay (C, magnification × 400) as described in Materials and Methods. ( D ) The protein levels of MMP2, MMP9, E-cadherin, N-cadherin, snail, slug, and GAPDH were detected with Western blotting analysis, and the pro and active forms of MMP2/9 were observed using gelatin zymography assay as described in Materials and Methods. ( E ) The mRNA levels of PLCG1, MMP2, MMP9, CDHI, CDH2, SNAIL, SLUG, and GAPDH were detected using Real-time PCR analysis as described in Materials and Methods. ( F ) The level of VEGF in extracellular matrix was detected using ELISA as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (* P

    Journal: Oncotarget

    Article Title: Lentivirus-mediated PLCγ1 gene short-hairpin RNA suppresses tumor growth and metastasis of human gastric adenocarcinoma

    doi: 10.18632/oncotarget.6976

    Figure Lengend Snippet: Lentivirus-mediated PLCγ1 shRNA could suppress migration in human gastric adenocarcinoma BGC-823 cells Cells were transduced with lentivirus-mediated PLCγ1 shRNA2/3 vectors. ( A ) The formation of membrane ruffles was detected using Ruffling assay as described in Materials and Methods. The cell nuclei were stained DAPI (blue) and the membrane ruffles were stained rhodamine-conjugated phalloidin (red). Scale bar = 10 μm. ( B and C ) The migration ability was measured using Transwell assay (B, magnification × 100) and Scratch assay (C, magnification × 400) as described in Materials and Methods. ( D ) The protein levels of MMP2, MMP9, E-cadherin, N-cadherin, snail, slug, and GAPDH were detected with Western blotting analysis, and the pro and active forms of MMP2/9 were observed using gelatin zymography assay as described in Materials and Methods. ( E ) The mRNA levels of PLCG1, MMP2, MMP9, CDHI, CDH2, SNAIL, SLUG, and GAPDH were detected using Real-time PCR analysis as described in Materials and Methods. ( F ) The level of VEGF in extracellular matrix was detected using ELISA as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (* P

    Article Snippet: PCNA, PARP, cleaved-PARP, MMP9, and GAPDH were purchased from Abcam (Cambridge, MA, USA).

    Techniques: shRNA, Migration, Transduction, Staining, Transwell Assay, Wound Healing Assay, Western Blot, Zymography Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Lentivirus-mediated PLCγ1 shRNA could block proliferation in human gastric adenocarcinoma BGC-823 cells BGC-823 cell line of stable expressing PLCγ1shRNA was established with the transduction of four types PLCγ1shRNAs using a lentiviral transduction strategy. ( A ) The effect of PLCγ1shRNAs on the level of PLCγ1 protein was detected with Western blotting analysis as described in Materials and Methods. ( B ) The effect of PLCγ1shRNAs on cell growth rate was measured with MTT assay as described in Materials and Methods. ( C ) The effect of PLCγ1 shRNA2/3 on cloning formation was detected with Colony formation assay as described in Materials and Methods. ( D ) The levels of PCNA, cleaved-PARP, PARP, Bcl-2, PLCγ1, and GAPDH protein were detected with Western blotting analysis as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (** P

    Journal: Oncotarget

    Article Title: Lentivirus-mediated PLCγ1 gene short-hairpin RNA suppresses tumor growth and metastasis of human gastric adenocarcinoma

    doi: 10.18632/oncotarget.6976

    Figure Lengend Snippet: Lentivirus-mediated PLCγ1 shRNA could block proliferation in human gastric adenocarcinoma BGC-823 cells BGC-823 cell line of stable expressing PLCγ1shRNA was established with the transduction of four types PLCγ1shRNAs using a lentiviral transduction strategy. ( A ) The effect of PLCγ1shRNAs on the level of PLCγ1 protein was detected with Western blotting analysis as described in Materials and Methods. ( B ) The effect of PLCγ1shRNAs on cell growth rate was measured with MTT assay as described in Materials and Methods. ( C ) The effect of PLCγ1 shRNA2/3 on cloning formation was detected with Colony formation assay as described in Materials and Methods. ( D ) The levels of PCNA, cleaved-PARP, PARP, Bcl-2, PLCγ1, and GAPDH protein were detected with Western blotting analysis as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (** P

    Article Snippet: PCNA, PARP, cleaved-PARP, MMP9, and GAPDH were purchased from Abcam (Cambridge, MA, USA).

    Techniques: shRNA, Blocking Assay, Expressing, Transduction, Western Blot, MTT Assay, Clone Assay, Colony Assay

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

    Journal: Oncology Letters

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

    doi: 10.3892/ol.2018.9391

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

    Article Snippet: Membranes were blocked in 5% bovine serum albumin (Sigma Aldrich; Merck KGaA) at room temperature for 1 h, and subsequently immunoblotted with the following primary antibodies according to the recommended dilution concentration: Anti-Ep3 (1:500; cat. no. P8372; Sigma-Aldrich; Merck KGaA), anti-caspase-3 (1:1,000; cat. no. 9662), anti-B cell lymphoma (Bcl)-2 (1:1,000; cat. no. 2872), anti-Bcl-associated × protein (Bax; 1:1,000; cat. no. 2772; all Cell Signaling Technology, Inc., Danvers, MA, USA), anti-MMP-9 (1:500; cat. no. ab58803; Abcam, Cambridge, UK), anti-VEGF (1:1,000; cat. no. V6627; Sigma-Aldrich; Meck KGaA), anti-TGF-β1 (1:1,000; cat. no. SAB4502954; Sigma-Aldrich; Merck KGaA), anti-Smad-2 (1:1,000; cat. no. ab63576), anti-Smad-3 (1:2,000; cat. no. ab40854), anti-phosphorylated (p)-Smad2 (1:800; cat. no. ab53100), anti-p-Smad3 (1:2,000; cat. no. ab52903) and anti-GAPDH (1:2,000; cat. no. ab8245; all Abcam) at 4°C overnight.

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

    GM1-deficient C6 cells are sensitized to CT by inhibition of sialylation or GSL biosynthesis. (A-E) C6 cells were cultured with the indicated inhibitors for 72 h followed by: (A) Staining was then performed with biotin-CTB, followed by DTAF-streptavidin. Fluorescence was measured by flow cytometry, represented here by MFI. (B) 1 h exposure to CT after which accumulated cAMP was measured by the cAMP-Glo™ luminescence assay. Luminescence signal is inversely proportional to cAMP levels. (C) As in panel A , but stained with biotin-PNA, followed by DTAF-streptavidin (D) Cell lysates were separated by PAGE and probed with biotin-PNA, biotin-CTB, or no biotinylated reagent, followed by streptavidin-peroxidase conjugate and development with chemiluminescent substrate. Equivalent amounts of protein were loaded in each lane and blots were probed with an anti-α-tubulin or anti-GAPDH antibody to confirm equivalent loading. (E) As in panel B , but brefeldin A (BFA) was added 1 h prior to CT addition and was also present during CT induction.

    Journal: PLoS Pathogens

    Article Title: GM1 ganglioside-independent intoxication by Cholera toxin

    doi: 10.1371/journal.ppat.1006862

    Figure Lengend Snippet: GM1-deficient C6 cells are sensitized to CT by inhibition of sialylation or GSL biosynthesis. (A-E) C6 cells were cultured with the indicated inhibitors for 72 h followed by: (A) Staining was then performed with biotin-CTB, followed by DTAF-streptavidin. Fluorescence was measured by flow cytometry, represented here by MFI. (B) 1 h exposure to CT after which accumulated cAMP was measured by the cAMP-Glo™ luminescence assay. Luminescence signal is inversely proportional to cAMP levels. (C) As in panel A , but stained with biotin-PNA, followed by DTAF-streptavidin (D) Cell lysates were separated by PAGE and probed with biotin-PNA, biotin-CTB, or no biotinylated reagent, followed by streptavidin-peroxidase conjugate and development with chemiluminescent substrate. Equivalent amounts of protein were loaded in each lane and blots were probed with an anti-α-tubulin or anti-GAPDH antibody to confirm equivalent loading. (E) As in panel B , but brefeldin A (BFA) was added 1 h prior to CT addition and was also present during CT induction.

    Article Snippet: Blots were incubated in mild stripping buffer (200 mM glycine, 0.1% (w/v) SDS, 1% (v/v) Tween-20, pH 2.2) at 37°C for 45 min and incubated with anti-histone H3 antibody (Abcam, Cat. No. ab1791, 1:2000 dilution, for AAL blot), anti-α-tubulin antibody (Sigma-Aldrich, Cat. No. T6199, 1:10000 dilution, for PNA and CTB blot), or anti-GAPDH antibody (Abcam, Cat. No. ab8245, 1:10000 dilution, for streptavidin-POD blot) at room temperature for 1 h and washed with TBST.

    Techniques: Inhibition, Cell Culture, Staining, CtB Assay, Fluorescence, Flow Cytometry, Cytometry, Luminescence Assay, Polyacrylamide Gel Electrophoresis

    Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Journal: The EMBO Journal

    Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy

    doi: 10.15252/embj.201899576

    Figure Lengend Snippet: Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

    Techniques: Activation Assay, Mouse Assay

    Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Journal: The EMBO Journal

    Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy

    doi: 10.15252/embj.201899576

    Figure Lengend Snippet: Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

    Techniques: Activation Assay, Mouse Assay

    Regulation of the expression of metastasis-related genes by Akt isoforms. GEO cells expressing non-targeting shRNA (sh) or Akt1, Akt2 or Akt3 shRNA were treated with Dox for 5 days and cDNA samples were subjected to real-time PCR using the Human Tumor Metastasis RT 2 Profiler PCR array from Super Array Biosciences (Qiagen). Top panel: Venn diagram showing differentially expressed metastatic genes on knockdown of each of the three Akt isoforms. Genes shown in red have higher expression in Akt isoform knockdown cells relative to non-targeting shRNA (sh) expressing cells, whereas genes in green show reduced expression. Lower panel: list of genes that showed differential expression ( > two-fold) on loss of individual Akt isoforms. MTSS1 is shown in bold.

    Journal: Oncogene

    Article Title: Role of Akt2 in regulation of metastasis suppressor 1 expression and colorectal cancer metastasis

    doi: 10.1038/onc.2016.460

    Figure Lengend Snippet: Regulation of the expression of metastasis-related genes by Akt isoforms. GEO cells expressing non-targeting shRNA (sh) or Akt1, Akt2 or Akt3 shRNA were treated with Dox for 5 days and cDNA samples were subjected to real-time PCR using the Human Tumor Metastasis RT 2 Profiler PCR array from Super Array Biosciences (Qiagen). Top panel: Venn diagram showing differentially expressed metastatic genes on knockdown of each of the three Akt isoforms. Genes shown in red have higher expression in Akt isoform knockdown cells relative to non-targeting shRNA (sh) expressing cells, whereas genes in green show reduced expression. Lower panel: list of genes that showed differential expression ( > two-fold) on loss of individual Akt isoforms. MTSS1 is shown in bold.

    Article Snippet: Two-step quantitative real-time PCR using TaqMan reagents and primers (Akt1: HS00178289_m1; Akt2: HS01086102_m1; Akt3: HS00987350_m1; Human GAPDH: 4352665; Mouse GAPDH: Mm99999915_g1) was performed according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). mRNA expression was normalized to levels of GAPDH.

    Techniques: Expressing, shRNA, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    HGF stimulation is abrogated by IQSEC1 depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and GAPDH (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: HGF stimulation is abrogated by IQSEC1 depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and GAPDH (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Western Blot, Expressing, shRNA, Quantitation Assay

    IQSEC1 isoforms differentially regulate collective invasion. (A) Schema, domain structure of IQSEC1 variants (v) 1-4. All variants possess an IQ motif, SEC7 and PH domains. v1 and v2 contain N-terminal extensions. A nuclear localization signal (NLS) is present in v1-3, lost in v4. v2/v3 share a common C-terminus. Common domains shown in grey, unique domains indicated by colour; blue; v1, pink; v2 and v3, green; v4. (B) Western blot in 2D and 3D using anti-IQSEC1 antibody. GAPDH is shown for IQSEC1 blot. Relative expression of all IQSEC1 bands normalised to 2D RWPE1 is shown. n=6 independent experiments. Values, mean ± s.d. p-values (Student’s t-test), *p ≤0.05; n.s. not significant. (C) Cartoon, isoform specific IQSEC1 functions were identified by comparing the growth and invasion of acini expressing different GFP-IQSEC1 variants upon depletion of endogenous IQSEC1 (shRNA KD4). (D) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4, and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, GFP and GAPDH antibodies. All antibodies used on same membrane. GAPDH is shown for IQSEC1 blot. Different exposures of IQSEC1 (long, short) are presented to demonstrate expression of all variants. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (E) Phase images of acini from cells described in D. GFP positive acini are outlined in yellow. Scale bars, 100μm. (F) Quantitation of images shown in E. Heatmap shows area and compactness measurements as Z-score-normalised to control (+-+-) values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower panels). n=3, 3 replicates/condition, 300-650 acini per condition. (G) PC3 acini described in D were stained for F-actin and nuclei (black and magenta respectively in upper panels) after 4 days. Green arrowheads, protrusions. Scale bars, 20μm. Localisation of GFP-IQSEC1 can be appreciated from FIRE pseudo coloured Look Up Table (FIRE LUT) (middle panels). Magnified images of boxed regions are inset. Cartoon, localization of GFP-IQSEC1 variants in PC3 acini (lower panel). (H) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4 using anti-IQSEC1 (specific for v2) and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. Different exposures of GFP (long, short) are presented showing GFP-IQSEC1 proteins. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (I) Schema, summary of the effect of IQSEC1 KD and expression of GFP-IQSEC1 v2 on growth and protrusive ability of PC3 acini. (J) Endogenous IQSEC1 v2 co-stained with F-actin (green and red respectively in merge). Magnified images of boxed region are shown. Red arrowhead indicates localisation at protrusion tip.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 isoforms differentially regulate collective invasion. (A) Schema, domain structure of IQSEC1 variants (v) 1-4. All variants possess an IQ motif, SEC7 and PH domains. v1 and v2 contain N-terminal extensions. A nuclear localization signal (NLS) is present in v1-3, lost in v4. v2/v3 share a common C-terminus. Common domains shown in grey, unique domains indicated by colour; blue; v1, pink; v2 and v3, green; v4. (B) Western blot in 2D and 3D using anti-IQSEC1 antibody. GAPDH is shown for IQSEC1 blot. Relative expression of all IQSEC1 bands normalised to 2D RWPE1 is shown. n=6 independent experiments. Values, mean ± s.d. p-values (Student’s t-test), *p ≤0.05; n.s. not significant. (C) Cartoon, isoform specific IQSEC1 functions were identified by comparing the growth and invasion of acini expressing different GFP-IQSEC1 variants upon depletion of endogenous IQSEC1 (shRNA KD4). (D) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4, and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, GFP and GAPDH antibodies. All antibodies used on same membrane. GAPDH is shown for IQSEC1 blot. Different exposures of IQSEC1 (long, short) are presented to demonstrate expression of all variants. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (E) Phase images of acini from cells described in D. GFP positive acini are outlined in yellow. Scale bars, 100μm. (F) Quantitation of images shown in E. Heatmap shows area and compactness measurements as Z-score-normalised to control (+-+-) values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower panels). n=3, 3 replicates/condition, 300-650 acini per condition. (G) PC3 acini described in D were stained for F-actin and nuclei (black and magenta respectively in upper panels) after 4 days. Green arrowheads, protrusions. Scale bars, 20μm. Localisation of GFP-IQSEC1 can be appreciated from FIRE pseudo coloured Look Up Table (FIRE LUT) (middle panels). Magnified images of boxed regions are inset. Cartoon, localization of GFP-IQSEC1 variants in PC3 acini (lower panel). (H) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4 using anti-IQSEC1 (specific for v2) and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. Different exposures of GFP (long, short) are presented showing GFP-IQSEC1 proteins. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (I) Schema, summary of the effect of IQSEC1 KD and expression of GFP-IQSEC1 v2 on growth and protrusive ability of PC3 acini. (J) Endogenous IQSEC1 v2 co-stained with F-actin (green and red respectively in merge). Magnified images of boxed region are shown. Red arrowhead indicates localisation at protrusion tip.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Western Blot, Expressing, shRNA, Quantitation Assay, Staining

    IQSEC1 regulates growth and invasion in multiple prostate cancer cell lines. (A) Heatmap shows mRNA expression levels, mined from CCLE, of the Met-PI3K-Akt pathway across different prostate cancer cell lines. (B) Western blot of prostate cancer cell lines using anti-IQSEC1, phospho-Y1234/1234 Met, total Met, LRP1, ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. (C) Schema, effect of NAV-2729 on ARF GTPase cycle. (D) Phase images of acini (+ and – HGF) at different time points are shown. Acini were also treated with NAV-2729 for 96 hours. Scale bars, 100μm. (E) Schema, summarizes the effect of each treatment described in D on acini growth and invasion. (F-I) Quantitation of 22Rv1 (F) , LNCaP (G) , DU145 (H) and DU145 + HGF (I) acini formation in the absence or presence of NAV-2729. 22Rv1 and DU145 acini were also expressing LRP1-GFP and IQSEC1-GFP v2 respectively. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2, replicates/condition, 400-1600 acini/condition.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 regulates growth and invasion in multiple prostate cancer cell lines. (A) Heatmap shows mRNA expression levels, mined from CCLE, of the Met-PI3K-Akt pathway across different prostate cancer cell lines. (B) Western blot of prostate cancer cell lines using anti-IQSEC1, phospho-Y1234/1234 Met, total Met, LRP1, ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. (C) Schema, effect of NAV-2729 on ARF GTPase cycle. (D) Phase images of acini (+ and – HGF) at different time points are shown. Acini were also treated with NAV-2729 for 96 hours. Scale bars, 100μm. (E) Schema, summarizes the effect of each treatment described in D on acini growth and invasion. (F-I) Quantitation of 22Rv1 (F) , LNCaP (G) , DU145 (H) and DU145 + HGF (I) acini formation in the absence or presence of NAV-2729. 22Rv1 and DU145 acini were also expressing LRP1-GFP and IQSEC1-GFP v2 respectively. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2, replicates/condition, 400-1600 acini/condition.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Expressing, Western Blot, Quantitation Assay

    IQSEC1 interacts with multiple transmembrane proteins. (A) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-IQSEC1, LRP1, Met, SORL1, RICTOR, Sin1, ARFGAP1, 14-3-3ζ/Δ, PPC6, GFP and GAPDH (shown for GFP blot) antibodies. (B) Western blot of PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (C) Western blot of Epi and EMT14 PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (D) Met immunoprecipitation was carried out on PC3 cells expressing Scr or IQSEC1 KD4 shRNA. Western blot was then performed on lysates (upper panels) and IPs (lower panels) using anti-LRP1, Met, IQSEC1 and GAPDH (shown for IQSEC1 blot) antibodies. Quantitation of LRP1/Met interaction normalised to Scr is shown. Values, mean ± s.d. n=3 independent experiments. p values (one-way ANOVA): n.s. not significant. (E-F) Western blot analysis of PC3 cells expressing Scr or (E) LRP1 and (F) Met shRNA. GAPDH is shown for Akt blots. (G) Quantitation of shape classification of PC3 cells expressing shRNAs for IQSEC1 binding partners as log2 fold change of each phenotype over control (left heatmap). p-values (one-way ANOVA): greyscale values as indicated (right heatmap). n=2, 4 replicates per condition, minimum 14,000 cells imaged/condition. (H) Quantitation of PC3 acini expressing GFP or LRP1-GFP. Heatmaps for area and compactness measurements shown as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 3 replicates, minimum 150 acini/condition. (I) Western blot of PC3 cells expressing SORL1 shRNA. GAPDH is shown for Akt blots. (J) Representative phase contrast images of PC3 acini expressing Scr (also shown in Figure 4H ), SORL1_ 2 and RICTOR_2 shRNA taken after 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum 700 acini/condition. (L-N) Western blots of PC3 cells expressing (L) RICTOR or (M) SIN1 or (N) ARFGAP1 shRNA. GAPDH is shown for Akt blots. (O) Representative phase contrast images of PC3 acini expressing Scr, ARFGAP1_1 and ARFGAP1_2 shRNA taken after 96 hours. Scale bars, 100μm. (P) Quantitation of PC3 acini shown in O. n=2, 4 replicates, minimum 1,100 acini/condition. (Q) Cartoon, depicts representative acini phenotype upon depletion of ARFGAP1 and ARF GTPase cycle.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 interacts with multiple transmembrane proteins. (A) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-IQSEC1, LRP1, Met, SORL1, RICTOR, Sin1, ARFGAP1, 14-3-3ζ/Δ, PPC6, GFP and GAPDH (shown for GFP blot) antibodies. (B) Western blot of PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (C) Western blot of Epi and EMT14 PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (D) Met immunoprecipitation was carried out on PC3 cells expressing Scr or IQSEC1 KD4 shRNA. Western blot was then performed on lysates (upper panels) and IPs (lower panels) using anti-LRP1, Met, IQSEC1 and GAPDH (shown for IQSEC1 blot) antibodies. Quantitation of LRP1/Met interaction normalised to Scr is shown. Values, mean ± s.d. n=3 independent experiments. p values (one-way ANOVA): n.s. not significant. (E-F) Western blot analysis of PC3 cells expressing Scr or (E) LRP1 and (F) Met shRNA. GAPDH is shown for Akt blots. (G) Quantitation of shape classification of PC3 cells expressing shRNAs for IQSEC1 binding partners as log2 fold change of each phenotype over control (left heatmap). p-values (one-way ANOVA): greyscale values as indicated (right heatmap). n=2, 4 replicates per condition, minimum 14,000 cells imaged/condition. (H) Quantitation of PC3 acini expressing GFP or LRP1-GFP. Heatmaps for area and compactness measurements shown as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 3 replicates, minimum 150 acini/condition. (I) Western blot of PC3 cells expressing SORL1 shRNA. GAPDH is shown for Akt blots. (J) Representative phase contrast images of PC3 acini expressing Scr (also shown in Figure 4H ), SORL1_ 2 and RICTOR_2 shRNA taken after 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum 700 acini/condition. (L-N) Western blots of PC3 cells expressing (L) RICTOR or (M) SIN1 or (N) ARFGAP1 shRNA. GAPDH is shown for Akt blots. (O) Representative phase contrast images of PC3 acini expressing Scr, ARFGAP1_1 and ARFGAP1_2 shRNA taken after 96 hours. Scale bars, 100μm. (P) Quantitation of PC3 acini shown in O. n=2, 4 replicates, minimum 1,100 acini/condition. (Q) Cartoon, depicts representative acini phenotype upon depletion of ARFGAP1 and ARF GTPase cycle.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Immunoprecipitation, Expressing, Western Blot, shRNA, Quantitation Assay, Binding Assay

    IQSEC1 activates ARF5/6 in distinct locations within protrusions. (A) Western blot of PC3 cells expressing Scr, ARF5 or ARF6 shRNA, alone or together, using anti ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. ARF intensity normalised to Scr is quantified. n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA). (B) Phase images of PC3 acini described in A. Yellow outlines indicate shRNA (mVenus) positive acini. Scale bars, 100μm. (C) Quantitation of images shown in B. Quantitation is shown for area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 5 replicates/condition, 2880 - 3188 acini quantified/condition. Cartoon, acini phenotype representative of each condition. (D) Western blot of PC3 cells stably co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) (Control) or ARF5-mNG and ARF6-RFP (ARF5/6), and either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH antibodies were used. Both endogenous and exogenous (OX) ARFs were detected. GAPDH is shown for ARF5 blot. (E) Phase images of PC3 acini described in D. Scale bars, 100μm. (F) Quantitation of images shown in E. n=2, 4 replicates/condition, 1,254 – 1,567 acini/condition. (G) PC3 cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2 and merged image of spindle shaped cell shown (left panel). Magnified images of boxed region are shown. White arrowheads indicate areas of colocalization. Scale bars, 20μm. (H) PC3 acini expressing either ARF5-mNG or ARF6-mNG were stained for IQSEC1. Localisation of these proteins is shown using FIRE LUT. Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. White arrows, lack of colocalization. Scale bars, 5μm. (I) Schema, GTPase cycle of ARF5 and ARF6, site of action of the IQSEC1-inhibiting molecules NAV-2729, the pan ARFGAP-inhibitor QS11, and GTP-loaded ARF detection by a GGA1-NGAT probe. (J) PC3 acini expressing either ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections (upper panels) and a single Z-slice of boxed regions (lower panels) shown. White and green arrowheads, colocalization in protrusions, white arrows, colocalization in cell body. Scale bars, 10μm. (K-L) PC3 cells expressing (K) ARF5-NG and GGA1-NGAT or (L) ARF6-NG and GGA1-NGAT were stably transfected with either Scr or IQSEC1 KD4 shRNA. Cells were treated with NAV-2729 or QS11 for 24 hours prior to fixation. Quantitation of % overlap of ARF and ARF-GTP probe per cell is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 3 replicates/condition with a minimum of 500 cells/condition. p-values (one-way ANOVA): *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. (M) Schema, different localization of IQSEC1 and active ARFs in protrusions is shown.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 activates ARF5/6 in distinct locations within protrusions. (A) Western blot of PC3 cells expressing Scr, ARF5 or ARF6 shRNA, alone or together, using anti ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. ARF intensity normalised to Scr is quantified. n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA). (B) Phase images of PC3 acini described in A. Yellow outlines indicate shRNA (mVenus) positive acini. Scale bars, 100μm. (C) Quantitation of images shown in B. Quantitation is shown for area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 5 replicates/condition, 2880 - 3188 acini quantified/condition. Cartoon, acini phenotype representative of each condition. (D) Western blot of PC3 cells stably co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) (Control) or ARF5-mNG and ARF6-RFP (ARF5/6), and either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH antibodies were used. Both endogenous and exogenous (OX) ARFs were detected. GAPDH is shown for ARF5 blot. (E) Phase images of PC3 acini described in D. Scale bars, 100μm. (F) Quantitation of images shown in E. n=2, 4 replicates/condition, 1,254 – 1,567 acini/condition. (G) PC3 cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2 and merged image of spindle shaped cell shown (left panel). Magnified images of boxed region are shown. White arrowheads indicate areas of colocalization. Scale bars, 20μm. (H) PC3 acini expressing either ARF5-mNG or ARF6-mNG were stained for IQSEC1. Localisation of these proteins is shown using FIRE LUT. Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. White arrows, lack of colocalization. Scale bars, 5μm. (I) Schema, GTPase cycle of ARF5 and ARF6, site of action of the IQSEC1-inhibiting molecules NAV-2729, the pan ARFGAP-inhibitor QS11, and GTP-loaded ARF detection by a GGA1-NGAT probe. (J) PC3 acini expressing either ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections (upper panels) and a single Z-slice of boxed regions (lower panels) shown. White and green arrowheads, colocalization in protrusions, white arrows, colocalization in cell body. Scale bars, 10μm. (K-L) PC3 cells expressing (K) ARF5-NG and GGA1-NGAT or (L) ARF6-NG and GGA1-NGAT were stably transfected with either Scr or IQSEC1 KD4 shRNA. Cells were treated with NAV-2729 or QS11 for 24 hours prior to fixation. Quantitation of % overlap of ARF and ARF-GTP probe per cell is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 3 replicates/condition with a minimum of 500 cells/condition. p-values (one-way ANOVA): *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. (M) Schema, different localization of IQSEC1 and active ARFs in protrusions is shown.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Western Blot, Expressing, shRNA, Quantitation Assay, Stable Transfection, Staining, Transfection

    IQSEC1-LRP1 complex regulates Met endocytic trafficking. (A) Western blot of PC3 cells co-expressing mNG and RFP (Control) or ARF5-mNG and ARF6-RFP (ARF5/6) with either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, and GAPDH (sample control) antibodies were used. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. n=2 independent experiments. Values, mean ± s.d. (C) Schema, effect of IQSEC1 on Met trafficking. (D) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were incubated with a Met-647 fluorescent antibody at 4°C (Surface) prior to stimulation with HGF for either 10 or 30 minutes (Internalisation). PC3 cells were also treated with chloroquine to allow accumulation of surface-derived Met (black) for 1 hour at 17°C prior to stimulation with HGF for either 10 or 30 minutes (Recycling). Cells were stained with F-actin (green outlines) and Hoechst (nuclei in magenta). Magnified images of boxed regions are inset. Scale bars 20µm. (E) Intensity of Met antibody in each cell was quantified (Z-score normalised); shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (F) Intensity of Met antibody in membrane, cytoplasmic and juxtanuclear regions was quantified. Line graphs show relative region intensities compared to the intensity of the whole cell (Z-score normalised). n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (G) Schema, sub-cellular re-localisation of active Met upon HGF treatment. (H) PC3 cells expressing Scr, IQSEC1 KD4, LRP1 or SORL1 shRNA were stimulated with HGF for 30 minutes. Cells were stained for phospho-Met (black), F-actin (green outlines) and Hoechst/nuclei (magenta). Magnified images of boxed regions are shown (lower panels). Cartoon, sub-cellular localization of active Met under different conditions. (I-J) Quantitation of spot intensity (Z-score normalised) is shown for images in H in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p-values (one-way ANOVA). (K) Schema, regulation of Met internalisation, but not recycling, by IQSEC1 and LRP1. All p values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1-LRP1 complex regulates Met endocytic trafficking. (A) Western blot of PC3 cells co-expressing mNG and RFP (Control) or ARF5-mNG and ARF6-RFP (ARF5/6) with either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, and GAPDH (sample control) antibodies were used. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. n=2 independent experiments. Values, mean ± s.d. (C) Schema, effect of IQSEC1 on Met trafficking. (D) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were incubated with a Met-647 fluorescent antibody at 4°C (Surface) prior to stimulation with HGF for either 10 or 30 minutes (Internalisation). PC3 cells were also treated with chloroquine to allow accumulation of surface-derived Met (black) for 1 hour at 17°C prior to stimulation with HGF for either 10 or 30 minutes (Recycling). Cells were stained with F-actin (green outlines) and Hoechst (nuclei in magenta). Magnified images of boxed regions are inset. Scale bars 20µm. (E) Intensity of Met antibody in each cell was quantified (Z-score normalised); shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (F) Intensity of Met antibody in membrane, cytoplasmic and juxtanuclear regions was quantified. Line graphs show relative region intensities compared to the intensity of the whole cell (Z-score normalised). n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (G) Schema, sub-cellular re-localisation of active Met upon HGF treatment. (H) PC3 cells expressing Scr, IQSEC1 KD4, LRP1 or SORL1 shRNA were stimulated with HGF for 30 minutes. Cells were stained for phospho-Met (black), F-actin (green outlines) and Hoechst/nuclei (magenta). Magnified images of boxed regions are shown (lower panels). Cartoon, sub-cellular localization of active Met under different conditions. (I-J) Quantitation of spot intensity (Z-score normalised) is shown for images in H in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p-values (one-way ANOVA). (K) Schema, regulation of Met internalisation, but not recycling, by IQSEC1 and LRP1. All p values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Western Blot, Expressing, shRNA, Quantitation Assay, Incubation, Derivative Assay, Staining

    IQSEC1 is a scaffold for Met signalling. (A) Schematic, IQSEC1 chimeras and variants display distinct phenotypes in 3D culture. GFP-trap immunoprecipitation was performed on PC3 cells expressing these proteins followed by tryptic digestion “on-beads” and LC/MS/MS analysis. IQSEC1 domain-specific interactions were identified and sorted by mRNA expression compared in paired PC3 subclones. (B) STRING network analysis of IQSEC1 binding partners identified by MS visualized using Cytoscape. Most IQSEC1 binding partners could be clustered into 8 protein complexes (colour coded). (C) Schema, indicates the protein complex each binding partner is associated with. Heatmap shows the fold change of IQSEC1 interactors from panel B binding to different IQSEC1 domains over GFP control. Values are −log 2 (FC), very high = −7 to −5, high = −5 to −2.5, medium −2.5 to −1, low = −1 to −0.05, no binding > 0.05. p-values are indicated in grey heatmap. The specific IQSEC1 domain to which each interactor binds is also depicted. Interactions were sorted according to the fold change in mRNA of non-invasive PC3 subclones (GS689.Li, EMT) compared to invasive subclones (PC3E, Epi) (RNAseq). (D) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-Met, LRP1, GFP, IQSEC1 and GAPDH antibodies. GAPDH is shown for LRP1 blot. Quantitation of LRP1/IQSEC1 and Met/IQSEC1 interactions for each GFP-trap are shown. n=3 and n=4, respectively. Values, mean ± s.d. p values (one-way ANOVA): **p≤0.01 and n.s. not significant. (E) Cartoon, depicts domain specific binding of IQSEC1 interactors. (F) PC3 acini expressing ARF5-mNG or ARF6-mNG were stained for Met and LRP1. FIRE LUT images are displayed with magnified images of boxed regions shown in lower panels. White arrowheads indicate localisation in protrusion tips. Scale bars, 5µm (upper) and 10µm (lower). (G) Schema, colocalization of IQSEC1-ARF interactors in protrusive tips. (H) Phase images of PC3 acini expressing Scr, LRP1_1, Met_2, SORL1_1, RICTOR_1 or SIN1_2 shRNA after 96 hours. Scale bars, 100μm. (I) Quantitation of images shown in H. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates/condition, 700-2,400 acini/condition. Cartoon, depicts acini phenotype representative of each condition.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1 is a scaffold for Met signalling. (A) Schematic, IQSEC1 chimeras and variants display distinct phenotypes in 3D culture. GFP-trap immunoprecipitation was performed on PC3 cells expressing these proteins followed by tryptic digestion “on-beads” and LC/MS/MS analysis. IQSEC1 domain-specific interactions were identified and sorted by mRNA expression compared in paired PC3 subclones. (B) STRING network analysis of IQSEC1 binding partners identified by MS visualized using Cytoscape. Most IQSEC1 binding partners could be clustered into 8 protein complexes (colour coded). (C) Schema, indicates the protein complex each binding partner is associated with. Heatmap shows the fold change of IQSEC1 interactors from panel B binding to different IQSEC1 domains over GFP control. Values are −log 2 (FC), very high = −7 to −5, high = −5 to −2.5, medium −2.5 to −1, low = −1 to −0.05, no binding > 0.05. p-values are indicated in grey heatmap. The specific IQSEC1 domain to which each interactor binds is also depicted. Interactions were sorted according to the fold change in mRNA of non-invasive PC3 subclones (GS689.Li, EMT) compared to invasive subclones (PC3E, Epi) (RNAseq). (D) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-Met, LRP1, GFP, IQSEC1 and GAPDH antibodies. GAPDH is shown for LRP1 blot. Quantitation of LRP1/IQSEC1 and Met/IQSEC1 interactions for each GFP-trap are shown. n=3 and n=4, respectively. Values, mean ± s.d. p values (one-way ANOVA): **p≤0.01 and n.s. not significant. (E) Cartoon, depicts domain specific binding of IQSEC1 interactors. (F) PC3 acini expressing ARF5-mNG or ARF6-mNG were stained for Met and LRP1. FIRE LUT images are displayed with magnified images of boxed regions shown in lower panels. White arrowheads indicate localisation in protrusion tips. Scale bars, 5µm (upper) and 10µm (lower). (G) Schema, colocalization of IQSEC1-ARF interactors in protrusive tips. (H) Phase images of PC3 acini expressing Scr, LRP1_1, Met_2, SORL1_1, RICTOR_1 or SIN1_2 shRNA after 96 hours. Scale bars, 100μm. (I) Quantitation of images shown in H. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates/condition, 700-2,400 acini/condition. Cartoon, depicts acini phenotype representative of each condition.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Immunoprecipitation, Expressing, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Western Blot, Quantitation Assay, Staining, shRNA

    Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Expressing, Transformation Assay, Activation Assay, Generated, Western Blot, Quantitation Assay

    IQSEC1-ARF signalling controls phosphoinositide generation in invasive protrusion tips. (A) Schema, signalling pathways in protrusive tips of PC3 acini. (B) PC3 acini expressing mNeonGreen tagged PH-PLCδ or PH-Grp1 were fixed after 3 days. PC3 acini were also stained with phospho-S473 Akt antibody. FIRE LUT used to show localisation and intensity of mNeonGreen or pAkt. Magnified images of boxed regions are also shown. White arrowheads, localisation at protrusive tips. Scale bars, 10 µm. Cartoon, spatial PIP production in protrusive tips. (C) Quantitation of cortical enrichment of PI(4,5)P 2 (upper panel) or PIP 3 per cell in the presence or absence of IQSEC1 is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, > 500 cells/condition. p-values (one-way ANOVA): ****p≤0.0001. (D) Cartoon, PIPK targeting in presence or absence of IQSEC1. (E) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control), Myr-FLAG-PIP5Kα, Myr-FLAG-PIP5Kβ or Myr-FLAG-PI3Kβ and either Scr or IQSEC1 shRNA (KD4). Anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies were used. GAPDH is shown for IQSEC1 blot. (F) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control) or Myr-Akt1 and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. (G) Phase images of PC3 acini described in E are shown. Scr acini were also treated with NAV-2729 (IQSEC1 inhibitor). Scale bars, 100μm. (H) Quantitation of images shown in G. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=3 with 4 replicates per condition. Between 1,693 and 2,435 acini quantified per condition. Cartoon, acini phenotype representative of each condition. (I) Phase images of PC3 acini described in F after 96 hours. Scr acini were also treated with IQSEC1-inhibiting compound NAV-2729. Scale bars, 100μm. (J) Quantitation of images shown in I. n=2 with 4 replicates/condition, 1,287-2,363 acini/condition. Cartoon, acini phenotype representative of each condition. (K) Schema, summarizes the relationship between location and level of cortical PIP 3 and 2D and 3D PC3 phenotype.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1-ARF signalling controls phosphoinositide generation in invasive protrusion tips. (A) Schema, signalling pathways in protrusive tips of PC3 acini. (B) PC3 acini expressing mNeonGreen tagged PH-PLCδ or PH-Grp1 were fixed after 3 days. PC3 acini were also stained with phospho-S473 Akt antibody. FIRE LUT used to show localisation and intensity of mNeonGreen or pAkt. Magnified images of boxed regions are also shown. White arrowheads, localisation at protrusive tips. Scale bars, 10 µm. Cartoon, spatial PIP production in protrusive tips. (C) Quantitation of cortical enrichment of PI(4,5)P 2 (upper panel) or PIP 3 per cell in the presence or absence of IQSEC1 is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, > 500 cells/condition. p-values (one-way ANOVA): ****p≤0.0001. (D) Cartoon, PIPK targeting in presence or absence of IQSEC1. (E) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control), Myr-FLAG-PIP5Kα, Myr-FLAG-PIP5Kβ or Myr-FLAG-PI3Kβ and either Scr or IQSEC1 shRNA (KD4). Anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies were used. GAPDH is shown for IQSEC1 blot. (F) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control) or Myr-Akt1 and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. (G) Phase images of PC3 acini described in E are shown. Scr acini were also treated with NAV-2729 (IQSEC1 inhibitor). Scale bars, 100μm. (H) Quantitation of images shown in G. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=3 with 4 replicates per condition. Between 1,693 and 2,435 acini quantified per condition. Cartoon, acini phenotype representative of each condition. (I) Phase images of PC3 acini described in F after 96 hours. Scr acini were also treated with IQSEC1-inhibiting compound NAV-2729. Scale bars, 100μm. (J) Quantitation of images shown in I. n=2 with 4 replicates/condition, 1,287-2,363 acini/condition. Cartoon, acini phenotype representative of each condition. (K) Schema, summarizes the relationship between location and level of cortical PIP 3 and 2D and 3D PC3 phenotype.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Expressing, Staining, Quantitation Assay, Western Blot, shRNA

    IQSEC1-ARF controls cortical PI(4,5)P 2 generation, which is required for production of PIP 3. (A) PC3 cells expressing mNeonGreen tagged PH-PLCδ (PIP 2 ) or PH-Grp1 (PIP 3 ) and either Scr or IQSEC1 shRNA KD4 were fixed after 2 days. FIRE LUT is used to show localisation and intensity of GFP in magnified images of boxed regions. White arrowhead indicates localisation at protrusive tip. Scale bars, 20µm. (B-C) Quantitation of (B) PI(4,5)P 2 or (C) PIP 3 cortical enrichment in spindle, spread and round cells expressing either Scr or IQSEC1 KD4 shRNA is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=3, 4 replicates/condition. 1330/1049 and 2222/1182 cells analysed for Src/ IQSEC1 KD4 shRNA respectively in B and C. p-values (one-way ANOVA). (D) Cartoon, summary of cortical PI(4,5)P 2 and PIP 3 localization in different cell shapes. (E) Western blot of PC3 cells treated with LY294002 (pan PI3K), AZD8835 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ), Cal-101 (PI3Kδ) and AktII (Akt) inhibitors for 24 hours. Anti-phospho-S473 Akt, Akt and GAPDH antibodies were used. (F) Phase contrast images of PC3 acini described in E. Scale bars, 100μm. (G) Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of PC3 acini shown in F. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmaps). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates, minimum of 350 acini/condition. (I) Western blot of PC3 cells expressing Scr, PIP5K1α or PIP5K1β shRNA using anti-PIP5K, IQSEC1, phospho-S473 Akt, Akt and GAPDH (shown for Akt blots) antibodies. (J) Phase contrast images of PC3 acini expressing Scr, PIP5K1α or PIP5K1β shRNA at 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum of 1,348 acini/condition. Cartoon, depicts acini phenotype representative of each condition. (L) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained for pS473 Akt (black) and Hoechst /nuclei (magenta). Magnified images of boxed regions are shown in lower panels. Blue arrowheads indicate localisation of active Akt. Scale bars, 10μm. (M-N) Quantitation of pAkt (M) intensity or (N) area in spots in the juxtanuclear, cytoplasm and periphery of PC3 cells expressing either Scr or IQSEC1 KD4 shRNA. Box-and-whiskers plots: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition. p values (Mann Whitney). (O) Cartoon, depicting phospho-Akt intensity, in the presence or absence of IQSEC1, at different subcellular locations. All p-values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Journal: bioRxiv

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    doi: 10.1101/851410

    Figure Lengend Snippet: IQSEC1-ARF controls cortical PI(4,5)P 2 generation, which is required for production of PIP 3. (A) PC3 cells expressing mNeonGreen tagged PH-PLCδ (PIP 2 ) or PH-Grp1 (PIP 3 ) and either Scr or IQSEC1 shRNA KD4 were fixed after 2 days. FIRE LUT is used to show localisation and intensity of GFP in magnified images of boxed regions. White arrowhead indicates localisation at protrusive tip. Scale bars, 20µm. (B-C) Quantitation of (B) PI(4,5)P 2 or (C) PIP 3 cortical enrichment in spindle, spread and round cells expressing either Scr or IQSEC1 KD4 shRNA is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=3, 4 replicates/condition. 1330/1049 and 2222/1182 cells analysed for Src/ IQSEC1 KD4 shRNA respectively in B and C. p-values (one-way ANOVA). (D) Cartoon, summary of cortical PI(4,5)P 2 and PIP 3 localization in different cell shapes. (E) Western blot of PC3 cells treated with LY294002 (pan PI3K), AZD8835 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ), Cal-101 (PI3Kδ) and AktII (Akt) inhibitors for 24 hours. Anti-phospho-S473 Akt, Akt and GAPDH antibodies were used. (F) Phase contrast images of PC3 acini described in E. Scale bars, 100μm. (G) Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of PC3 acini shown in F. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmaps). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates, minimum of 350 acini/condition. (I) Western blot of PC3 cells expressing Scr, PIP5K1α or PIP5K1β shRNA using anti-PIP5K, IQSEC1, phospho-S473 Akt, Akt and GAPDH (shown for Akt blots) antibodies. (J) Phase contrast images of PC3 acini expressing Scr, PIP5K1α or PIP5K1β shRNA at 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum of 1,348 acini/condition. Cartoon, depicts acini phenotype representative of each condition. (L) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained for pS473 Akt (black) and Hoechst /nuclei (magenta). Magnified images of boxed regions are shown in lower panels. Blue arrowheads indicate localisation of active Akt. Scale bars, 10μm. (M-N) Quantitation of pAkt (M) intensity or (N) area in spots in the juxtanuclear, cytoplasm and periphery of PC3 cells expressing either Scr or IQSEC1 KD4 shRNA. Box-and-whiskers plots: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition. p values (Mann Whitney). (O) Cartoon, depicting phospho-Akt intensity, in the presence or absence of IQSEC1, at different subcellular locations. All p-values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Article Snippet: Antibodies used were as follows; anti-GAPDH (CST 2118 1:5000), anti-IQSEC1 (Sigma G4798), anti-IQSEC1 (Caltag-Medsystems PSI-8009), anti-GFP (Merck 000000011814460001), anti-LRP1 (Sigma L2295), anti-Met (CST 3127), anti-Met phospho 1234/1235 (CST 3077), anti-Akt (CST 2920), anti-Akt phospho S473 (CST 3787), anti-ARF1 (Novus Biologicals NB-110-85530), anti-ARF6 (Sigma A5230), ARF5 (Novus Biologicals H00000381-M01), anti-ARFGAP1 (Sigma HPA051019), anti-SORL1 (BD 611860), anti-Sin1 (CST 12860), anti-RICTOR (CST 2114), anti-PPC6 (Sigma HPA050940 1:250), anti-14-3-3ζ/Δ (CST 7413), anti-PIP5Ka (CST 9693) and PIP5Kb (Sigma K0767).

    Techniques: Expressing, shRNA, Quantitation Assay, Western Blot, Staining, MANN-WHITNEY

    Quantitative real time polymerase chain reaction (qRT-PCR) of patient survivin levels. A: qRT-PCR was performed to compare survivin expression between tumor and adjacent squamous epithelial tissue. B: On average, tumor samples showed 3× greater survivin expression than paired adjacent tissue.

    Journal: PLoS ONE

    Article Title: Prognostic Value and Targeted Inhibition of Survivin Expression in Esophageal Adenocarcinoma and Cancer-Adjacent Squamous Epithelium

    doi: 10.1371/journal.pone.0078343

    Figure Lengend Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) of patient survivin levels. A: qRT-PCR was performed to compare survivin expression between tumor and adjacent squamous epithelial tissue. B: On average, tumor samples showed 3× greater survivin expression than paired adjacent tissue.

    Article Snippet: PCR was performed in a total volume of 20 ul using 600 ng cDNA, 1× Taqman PCR universal mastermix (Invitrogen, Grand Island, NY, 4304437), and 1× Survivin or GAPDH primer (Applied Biosystems, Carlsbad, CA hs03043576_m1 and hs99999905_m1) for each reaction.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Effect of G17 on Snail expression in gastric cancer cells . (A) AGSE cells were treated as in 1A or (B) 1B above and subjected to Western Blot analysis utilizing antibodies against Snail and GAPDH (as control). (C) Western Blot analysis of cell extracts with the indicated antibodies, treated with 100 nM G17 for 1 hour, following an overnight pretreatment with 100 nM YM 022. (D) Subconfluent AGSE cells were transiently transfected with Snail-luciferase vector (Snail-luc) along with β-Gal vector (for normalization of transfection). Forty-eight hours after transfection, cells were treated overnight in the presence (+) or absence (-) of 100 nM G17, and luciferase and β-Gal assays were performed. The RLU/β-Gal values were represented as percent control, considering the untreated samples as 100%. Each transfection was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments.

    Journal: Journal of Molecular Signaling

    Article Title: Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells

    doi: 10.1186/1750-2187-5-9

    Figure Lengend Snippet: Effect of G17 on Snail expression in gastric cancer cells . (A) AGSE cells were treated as in 1A or (B) 1B above and subjected to Western Blot analysis utilizing antibodies against Snail and GAPDH (as control). (C) Western Blot analysis of cell extracts with the indicated antibodies, treated with 100 nM G17 for 1 hour, following an overnight pretreatment with 100 nM YM 022. (D) Subconfluent AGSE cells were transiently transfected with Snail-luciferase vector (Snail-luc) along with β-Gal vector (for normalization of transfection). Forty-eight hours after transfection, cells were treated overnight in the presence (+) or absence (-) of 100 nM G17, and luciferase and β-Gal assays were performed. The RLU/β-Gal values were represented as percent control, considering the untreated samples as 100%. Each transfection was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments.

    Article Snippet: The antibodies utilized were obtained from the following sources: GSK3β, pGSK3βSer9 , AKT, pAKTSer473 , pc-Jun, c-Jun, Snail and Lamin A/C (Cell Signaling Technology, Danvers, MA), GAPDH (Ambion, Austin, TX), β-catenin (BD Biosciences, San Jose, CA), HA.11 (Covance, Berkeley, CA).

    Techniques: Expressing, Western Blot, Transfection, Luciferase, Plasmid Preparation

    Effect of GSK3β inhibition on G17-induced Snail expression and β-catenin nuclear translocation . (A) AGSE cells were treated with (+) or without (-) 100 nM G17, following an overnight pretreatment with either none (lanes 1, 2), 5 μM (lanes 3, 4) or 10 μM (lanes 5, 6) AR-A014418. Western Blot analysis was then performed with the antibodies indicated. (B) Luciferase (with Snail-luc) and β-Gal assays were performed as in 2D following a 1 hour pretreatment with AR-A014418. (C) AGSE cells were co-transfected with Snail-luc and β-Gal vectors along with either Empty vector (lanes 1, 2), GSK3β-S9A mutant vector (lanes 3, 4) or GSK3β-K/A mutant vector (lanes 5, 6). Luciferase and β-Gal assays were performed after G17 treatment as in 2D. Each transfection (3B, 3C) was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments. (D) Upper Panel : Confluent AGSE cells were treated with G17 for 8 hours after an overnight pretreatment with none (lanes 1, 2), or AR-A014418 (lanes 3, 4) or SP600125 (lanes 5, 6). At the end of treatment, nuclear protein was isolated and subjected to Western Blot analysis with antibodies against β-catenin, GAPDH (cytoplasmic protein) or Lamin A/C (nuclear protein). Lower Panel : Cells were pretreated as in the upper panel, followed by 1 hour G17 treatment and Western Blot analysis.

    Journal: Journal of Molecular Signaling

    Article Title: Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells

    doi: 10.1186/1750-2187-5-9

    Figure Lengend Snippet: Effect of GSK3β inhibition on G17-induced Snail expression and β-catenin nuclear translocation . (A) AGSE cells were treated with (+) or without (-) 100 nM G17, following an overnight pretreatment with either none (lanes 1, 2), 5 μM (lanes 3, 4) or 10 μM (lanes 5, 6) AR-A014418. Western Blot analysis was then performed with the antibodies indicated. (B) Luciferase (with Snail-luc) and β-Gal assays were performed as in 2D following a 1 hour pretreatment with AR-A014418. (C) AGSE cells were co-transfected with Snail-luc and β-Gal vectors along with either Empty vector (lanes 1, 2), GSK3β-S9A mutant vector (lanes 3, 4) or GSK3β-K/A mutant vector (lanes 5, 6). Luciferase and β-Gal assays were performed after G17 treatment as in 2D. Each transfection (3B, 3C) was performed in triplicate, and the data represent the mean ± SD of at least two independent experiments. (D) Upper Panel : Confluent AGSE cells were treated with G17 for 8 hours after an overnight pretreatment with none (lanes 1, 2), or AR-A014418 (lanes 3, 4) or SP600125 (lanes 5, 6). At the end of treatment, nuclear protein was isolated and subjected to Western Blot analysis with antibodies against β-catenin, GAPDH (cytoplasmic protein) or Lamin A/C (nuclear protein). Lower Panel : Cells were pretreated as in the upper panel, followed by 1 hour G17 treatment and Western Blot analysis.

    Article Snippet: The antibodies utilized were obtained from the following sources: GSK3β, pGSK3βSer9 , AKT, pAKTSer473 , pc-Jun, c-Jun, Snail and Lamin A/C (Cell Signaling Technology, Danvers, MA), GAPDH (Ambion, Austin, TX), β-catenin (BD Biosciences, San Jose, CA), HA.11 (Covance, Berkeley, CA).

    Techniques: Inhibition, Expressing, Translocation Assay, Western Blot, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Isolation

    Effect of overexpression of GSK3β on G17-induced migration . (A) . Subconfluent AGSE cells were transiently transfected with Empty Vector, GSK3β-WT, GSK3β-KA mutant or GSK3β-S9A mutant vectors. The cells were wounded linearly 48 hours post-transfection and, after an overnight recovery following wounding, they were treated with G17 and pictures obtained at the indicated times. (B) AGSE cells were transfected as in 4A followed by G17 treatment and wound healing assay. The distance of migration of the wounded edges for each time point were measured at several places and the average distance was represented by bar diagrams as

    Journal: Journal of Molecular Signaling

    Article Title: Glycogen Synthase Kinase-3beta regulates Snail and beta-catenin during gastrin-induced migration of gastric cancer cells

    doi: 10.1186/1750-2187-5-9

    Figure Lengend Snippet: Effect of overexpression of GSK3β on G17-induced migration . (A) . Subconfluent AGSE cells were transiently transfected with Empty Vector, GSK3β-WT, GSK3β-KA mutant or GSK3β-S9A mutant vectors. The cells were wounded linearly 48 hours post-transfection and, after an overnight recovery following wounding, they were treated with G17 and pictures obtained at the indicated times. (B) AGSE cells were transfected as in 4A followed by G17 treatment and wound healing assay. The distance of migration of the wounded edges for each time point were measured at several places and the average distance was represented by bar diagrams as "Average Gap". (C) AGSE cells transfected in A and treated with G17 were analyzed for protein expression. Western Blot analysis was performed with an HA.11 antibody to detect ectopic HA-tagged GSK3β proteins and with β-catenin and GAPDH antibodies to detect the corresponding endogenous proteins.

    Article Snippet: The antibodies utilized were obtained from the following sources: GSK3β, pGSK3βSer9 , AKT, pAKTSer473 , pc-Jun, c-Jun, Snail and Lamin A/C (Cell Signaling Technology, Danvers, MA), GAPDH (Ambion, Austin, TX), β-catenin (BD Biosciences, San Jose, CA), HA.11 (Covance, Berkeley, CA).

    Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Mutagenesis, Wound Healing Assay, Expressing, Western Blot

    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

    A short isoform of Sup35 appears during fermentative growth in glucose. A, GT17 [ psi − pin − ] cells grown in the mid log-phase ( lane 5 ) were starved in YPA medium for 12 h ( lane 6 ), and cultured in YPDA. Cells were harvested at the specified time points ( lanes 7–14 ) and analyzed by Western blotting ( WB ) using anti-Sup35 and anti-GAPDH. The four leftmost lanes indicate 2-fold dilutions of the protein to confirm that the condition used was semi-quantitative. B, the protein levels of Sup35 ( blue ) and Sup35s ( red ) were measured by MultiGauge and normalized to that of GAPDH, and the normalized Sup35 level (0 h) was defined as 100%. Dotted line indicates the A 600 of the yeast culture.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteolysis suppresses spontaneous prion generation in yeast

    doi: 10.1074/jbc.M117.811323

    Figure Lengend Snippet: A short isoform of Sup35 appears during fermentative growth in glucose. A, GT17 [ psi − pin − ] cells grown in the mid log-phase ( lane 5 ) were starved in YPA medium for 12 h ( lane 6 ), and cultured in YPDA. Cells were harvested at the specified time points ( lanes 7–14 ) and analyzed by Western blotting ( WB ) using anti-Sup35 and anti-GAPDH. The four leftmost lanes indicate 2-fold dilutions of the protein to confirm that the condition used was semi-quantitative. B, the protein levels of Sup35 ( blue ) and Sup35s ( red ) were measured by MultiGauge and normalized to that of GAPDH, and the normalized Sup35 level (0 h) was defined as 100%. Dotted line indicates the A 600 of the yeast culture.

    Article Snippet: The extracts were analyzed by Western blotting using the following antibodies; anti-Sup35 antibody raised against His-tagged Sup35 (136–483), anti-Prb1 antibody raised against His-tagged mature Prb1 (281–573), anti-GAPDH antibody raised against His-tagged human GAPDH (1–335), and anti-FLAG M2 antibody (Sigma).

    Techniques: Cell Culture, Western Blot

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

    Journal: Aging Cell

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

    doi: 10.1111/acel.12328

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

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

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

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

    Journal: Aging Cell

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

    doi: 10.1111/acel.12328

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

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

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

    Expression of STAT5b mRNA and protein in pancreatic cancer cells. (A) Quantitative RT-PCR analysis of STAT5b mRNA levels. STAT5b mRNA expressed in all pancreatic cancer cell lines, and the levels were highest in PANC-1 cells, second highest in Capan-1 cells, and lowest in PK-45H cells. In PANC-1 cells, STAT5b mRNA levels were ~13.5-fold higher than in PK-45H cells. (B) Western blot analysis of STAT5b protein levels. STAT5b protein was detected in all cell lines consistent with the RT-PCR results and the levels were highest in PANC-1 cells and second highest in Capan-1 cells.

    Journal: Oncology Reports

    Article Title: Suppression of STAT5b in pancreatic cancer cells leads to attenuated gemcitabine chemoresistance, adhesion and invasion

    doi: 10.3892/or.2016.4727

    Figure Lengend Snippet: Expression of STAT5b mRNA and protein in pancreatic cancer cells. (A) Quantitative RT-PCR analysis of STAT5b mRNA levels. STAT5b mRNA expressed in all pancreatic cancer cell lines, and the levels were highest in PANC-1 cells, second highest in Capan-1 cells, and lowest in PK-45H cells. In PANC-1 cells, STAT5b mRNA levels were ~13.5-fold higher than in PK-45H cells. (B) Western blot analysis of STAT5b protein levels. STAT5b protein was detected in all cell lines consistent with the RT-PCR results and the levels were highest in PANC-1 cells and second highest in Capan-1 cells.

    Article Snippet: Quantitative RT-PCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) and for evaluation, commercial TaqMan® Gene Expression- assays (Applied Biosystems, AoD, Assay-ID: Hs00273500_m1 for the studied genes STAT5b and Hs02758991_g1 for GAPDH as the reference gene) were used with optimized primer and probe concentrations.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Chromatin architecture of the Reg locus in adult cortex and pancreas. ( A ) Levels of cohesin were assayed by immunoblot with SA1 and Rad21 antibodies in pancreas from wild-type and SA1 heterozygous mice (two individuals per genotype). GAPDH serves as loading control. ( B ) Table showing the four genes differentially expressed (FDR

    Journal: Nucleic Acids Research

    Article Title: The contribution of cohesin-SA1 to gene expression and chromatin architecture in two murine tissues

    doi: 10.1093/nar/gkv144

    Figure Lengend Snippet: Chromatin architecture of the Reg locus in adult cortex and pancreas. ( A ) Levels of cohesin were assayed by immunoblot with SA1 and Rad21 antibodies in pancreas from wild-type and SA1 heterozygous mice (two individuals per genotype). GAPDH serves as loading control. ( B ) Table showing the four genes differentially expressed (FDR

    Article Snippet: Equal amounts of protein were run in 7.5 Bis/Tris gels followed by western blotting with antibodies against SA1 and Rad21 ( ) and GAPDH (Sigma, G8795).

    Techniques: Mouse Assay

    K nockdown of FTO does not significantly change mRNA levels of genes involved in miRNA biogenesis. The steady-state mRNA levels of DICER , DROSHA , DGCR8 and ADAR were analyzed by qRT-PCR in cells treated with scrambled (scr) and FTO- specific siRNAs, respectively. GAPDH was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. FTO kd, FTO -specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.

    Journal: PLoS ONE

    Article Title: N6-Adenosine Methylation in MiRNAs

    doi: 10.1371/journal.pone.0118438

    Figure Lengend Snippet: K nockdown of FTO does not significantly change mRNA levels of genes involved in miRNA biogenesis. The steady-state mRNA levels of DICER , DROSHA , DGCR8 and ADAR were analyzed by qRT-PCR in cells treated with scrambled (scr) and FTO- specific siRNAs, respectively. GAPDH was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. FTO kd, FTO -specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.

    Article Snippet: The primary antibodies used for western blotting were mouse and rabbit anti-FTO (Abcam, Cambridge, UK; Epitomics, Burlingame CA, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Quantitative RT-PCR

    FTO knockdown in HEK293 cell clones FTO1C1, FTO2D4 and FTO3C3. A) The levels of FTO transcripts were investigated by qRT-PCR. GAPDH was used as a reference gene. Mean ± SD for three pairs of scrambled (scr) and FTO specific siRNAs treated cells are shown. B) Reduced protein levels were revealed by Western blot in all three cell lines transfected with siRNAs targeting FTO mRNA. Exemplary photo is depicted.

    Journal: PLoS ONE

    Article Title: N6-Adenosine Methylation in MiRNAs

    doi: 10.1371/journal.pone.0118438

    Figure Lengend Snippet: FTO knockdown in HEK293 cell clones FTO1C1, FTO2D4 and FTO3C3. A) The levels of FTO transcripts were investigated by qRT-PCR. GAPDH was used as a reference gene. Mean ± SD for three pairs of scrambled (scr) and FTO specific siRNAs treated cells are shown. B) Reduced protein levels were revealed by Western blot in all three cell lines transfected with siRNAs targeting FTO mRNA. Exemplary photo is depicted.

    Article Snippet: The primary antibodies used for western blotting were mouse and rabbit anti-FTO (Abcam, Cambridge, UK; Epitomics, Burlingame CA, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Clone Assay, Quantitative RT-PCR, Western Blot, Transfection

    Deregulation of miRNAs in FTO knockdown cells. Mature miRNAs showing increased (A) and decreased (B) steady state levels in FTO knockdown cells. Normalized RNA-seq read numbers of individual miRNAs in FTO knockdown and scrambled siRNA treated cells were compared. Mean ± SD of three independent experiments are depicted. For verification and further studies, qRT-PCR analyses of selected mature miRNAs (C) and primary miRNA transcripts (D) were performed. We did not use other small RNAs as a reference gene for measuring mature miRNAs levels (as suggested by Life Technologies), since depletion of FTO might have an impact on their levels. Therefore, luciferase RNA was used to generate a standard curve and added to the qRT-PCR assays. GAPDH was used as a reference gene for measuring primary miRNAs transcript levels. Mean ± SD from quadruplicates per assay for three independent cell lines (FTO1C1, FTO2D4 and FTO3C3) are depicted. kd, FTO specific siRNA treated cells, scr, scrambled siRNA treated cells.

    Journal: PLoS ONE

    Article Title: N6-Adenosine Methylation in MiRNAs

    doi: 10.1371/journal.pone.0118438

    Figure Lengend Snippet: Deregulation of miRNAs in FTO knockdown cells. Mature miRNAs showing increased (A) and decreased (B) steady state levels in FTO knockdown cells. Normalized RNA-seq read numbers of individual miRNAs in FTO knockdown and scrambled siRNA treated cells were compared. Mean ± SD of three independent experiments are depicted. For verification and further studies, qRT-PCR analyses of selected mature miRNAs (C) and primary miRNA transcripts (D) were performed. We did not use other small RNAs as a reference gene for measuring mature miRNAs levels (as suggested by Life Technologies), since depletion of FTO might have an impact on their levels. Therefore, luciferase RNA was used to generate a standard curve and added to the qRT-PCR assays. GAPDH was used as a reference gene for measuring primary miRNAs transcript levels. Mean ± SD from quadruplicates per assay for three independent cell lines (FTO1C1, FTO2D4 and FTO3C3) are depicted. kd, FTO specific siRNA treated cells, scr, scrambled siRNA treated cells.

    Article Snippet: The primary antibodies used for western blotting were mouse and rabbit anti-FTO (Abcam, Cambridge, UK; Epitomics, Burlingame CA, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Luciferase

    P2X7 and EGF Receptor Stimulation Regulates the Expression of DUSP6 in Cerebellar Granule Neurons and Astrocytes ) for different times and DUSP6 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the total lysates by immunoblotting. (A) Immunoblots of a representative time-course experiment for each effector. (B) Cells were incubated with or without the effectors, 300 μM BzATP or 100 ng/mL EGF, for 30 min, fixed and the presence of DUSP6 was detected by immunocytochemistry. Representative immunofluorescence images are shown. Scale bars represent 50 μm. (C) The diagrams represent the quantification of the time courses obtained by normalization to the corresponding GAPDH levels. The data are represented as the means ± SEM from three independent experiments performed on different cultures. *** p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: P2X7 Nucleotide and EGF Receptors Exert Dual Modulation of the Dual-Specificity Phosphatase 6 (MKP-3) in Granule Neurons and Astrocytes, Contributing to Negative Feedback on ERK Signaling

    doi: 10.3389/fnmol.2017.00448

    Figure Lengend Snippet: P2X7 and EGF Receptor Stimulation Regulates the Expression of DUSP6 in Cerebellar Granule Neurons and Astrocytes ) for different times and DUSP6 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the total lysates by immunoblotting. (A) Immunoblots of a representative time-course experiment for each effector. (B) Cells were incubated with or without the effectors, 300 μM BzATP or 100 ng/mL EGF, for 30 min, fixed and the presence of DUSP6 was detected by immunocytochemistry. Representative immunofluorescence images are shown. Scale bars represent 50 μm. (C) The diagrams represent the quantification of the time courses obtained by normalization to the corresponding GAPDH levels. The data are represented as the means ± SEM from three independent experiments performed on different cultures. *** p

    Article Snippet: Antisera for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was generated in rabbit and purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing, Western Blot, Incubation, Immunocytochemistry, Immunofluorescence

    Atorvastatin effect on the expression of smooth muscle actin. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; SMA: Smooth muscle actin.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Atorvastatin and rosuvastatin do not prevent thioacetamide induced liver cirrhosis in rats

    doi: 10.3748/wjg.v19.i2.241

    Figure Lengend Snippet: Atorvastatin effect on the expression of smooth muscle actin. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; SMA: Smooth muscle actin.

    Article Snippet: Equal amounts of total protein-were separated in 4%-12% bis-tris (BT) gels (NuPAGE, Gibco-BRL Life Technologies, Grand Island, NY), blotted onto Hybond C extra membranes, blocked overnight in 5% milk, and incubated with antibodies against α smooth muscle actin (αSMA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, United States) and then incubated with horseradish peroxidase-conjugated secondary antibody.

    Techniques: Expressing