Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: RT-PCR analysis of PRL-3 mRNA expression levels in 19 bone marrow samples from AML patients either negative (ITD NEG; n = 12) or positive (ITD POS; n = 7) for FLT3-ITD mutation. MOLM-14 and MV4-11 AML cell lines were used as FLT3-ITD positive controls. β-actin, loading control. (a–d) Microarray data analysis of PRL-3 mRNA levels in FLT-ITD-positive (POS) or FLT3-ITD-negative (NEG) patients in four independent patient cohorts (total n = 1158). (a) Cohort 1 AML patient with normal karyotype ( n = 101, p = 0.001). (b) GSE1159 AML patient cohort ( n = 285, p < 0.001). (c) GSE6891 AML patient cohort ( n = 521, p < 0.001). (d) GSE15434 AML patient cohort ( n = 251, p < 0.001). Statistical differences between ITD-POS and ITD-NEG patients were determined using Chi-square test. PRL-3 expression level is divided into four groups: very high, high, intermediate, low. Western blot analysis of PRL-3 protein levels in four AML cell lines. Western blot analysis of PRL-3 in MOLM-14 and MV4-11 cells upon siRNA-mediated knock-down of FLT3 expression. NS, control non-silencing siRNA. GAPDH, loading control.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis, Microarray, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: A–C. Kaplan–Meier analysis of overall survival (OS) in normal karyotype AML patients for PRL-3 mRNA expression in (A) Cohort 1 AML patients, n = 101, (B) GSE6891, n = 227 and (C) GSE12417, n = 163. Statistically significant p values (using the log-rank test) are indicated in the figures.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: TF1-ITD and MOLM-14 cells were incubated with various concentrations of FLT3 inhibitors (PKC412, CEP-701) or Src inhibitors (SU6656, PP2) for 24 h. Whole cell lysates were subjected to Western blot analysis with indicated antibodies. GAPDH, loading control. (a–d) Western blot analysis of AML cells upon FLT3 inhibition. PKC412 and CEP-701 inhibited the phosphorylation of both FLT3 and STAT5 as well as PRL-3 protein levels in a dose-dependent manner in both TF1-ITD (a, b) and MOLM-14 (c, d) cells (a–d) Western blot analysis of AML cells upon FLT3 inhibition. PKC412 and CEP-701 inhibited the phosphorylation of Src, but not JAK, in a dose-dependent manner in both TF1-ITD (a, b) and MOLM-14 (c, d) cells. (a, b) Western blot analysis of AML cells upon Src inhibition. SU6656 (a) and PP2 (b) inhibited the phosphorylation of Src and STAT5 as well as PRL-3 protein levels in a dose-dependent manner in TF1-ITD cells.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Incubation, Western Blot, Inhibition

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: Two putative STAT5 binding sites (S1 and S2; DNA sequences illustrated) in a distal 5′-flanking region of PRL-3, as predicted by TRANSFAC. EMSA analysis using S1 and S2 biotinylated DNA probes (S1 and S2) incubated with nuclear extracts from either TF-1 or TF1-ITD cells. Arrow, shifted protein/probe complex. EMSA analysis as in (B) in the presence of 10-fold molar excess of unlabelled STAT5 competitor. Western blot analysis of streptavidin–agarose pull-down fractions (unbound or bound) using probe S1. Left panel: schematic diagram of a −5.4 kb upstream sequence of PRL-3 and its 5′-sequential deletion sequence with luciferase reporter vector (pGL3-S1a, S1b, S1c and S1d), respectively. Right panel: STAT5A or STAT5B expression vectors were co-transfected with PRL-3 luciferase reporter vector to TF-1 cells and luciferase activity measured. Error bars represent the mean ± SD from three independent experiments. PRL-3 expression is down-regulated upon siRNA-mediated STAT5 depletion in AML cells. NS, control non-silencing siRNA. (a) Quantitative real time PCR analysis of PRL-3 mRNA level after knock-down of STAT5 gene, normalized to GAPDH mRNA. Statistical differences between two groups were determined using Student's t -test (mean ± SD, n = 3). *** p < 0.001 (for TF1-ITD); ** p = 0.011 (for MOLM-14); * p = 0.038 (for MV4-11). (b) Western blot analysis of PRL-3 protein level after knock-down of STAT5 gene. GAPDH, loading control.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Binding Assay, Incubation, Western Blot, Sequencing, Luciferase, Plasmid Preparation, Expressing, Transfection, Activity Assay, Real-time Polymerase Chain Reaction

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: SEAP reporter assay results measuring AP-1 activity in TF-1 cells overexpressing GFP (TF1-GFP) or GFP-PRL-3 (TF1-PRL-3). Error bars represent the mean ± SD from three independent experiments. (a, b) PRL-3 specifically upregulates c-Jun but not c-Fos. (a) Western blot analysis of TF1-GFP, TF1-PRL-3 and TF1-ITD cells. (b) Western blot analysis after knock-down of endogenous PRL-3 in TF1-ITD cells. GAPDH, loading control. NS, control non-silencing siRNA. (a–d) PRL-3-mediated upregulation of c-Jun is dependent on ERK and JNK pathways. (a) Western blot analysis after knock-down of endogenous PRL-3 in TF1-ITD and MOLM-14 cells. (b) Western blot analysis of TF1-GFP and TF1-PRL-3 cells. (c, d) Western blot analysis after knock-down of ERK1/2 or JNK in TF1-PRL-3 cells. GAPDH, loading control. NS, control non-silencing siRNA. (a–c) MTS assay results reflecting numbers of viable TF1-PRL-3 cells after treatment with ERK-specific inhibitor (U0126), JNK-specific inhibitor (SP600125) or general AP-1 inhibitor (curcumin) for the various time points. Error bars represent the mean ± SD from three independent experiments.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Reporter Assay, Activity Assay, Western Blot, MTS Assay

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: Right panel: MTS assay results reflecting numbers of viable TF1-GFP and TF1-PRL-3 cells after culture in the absence of cytokines for various durations. Error bars represent the mean ± SD from three independent experiments. Left panel: Western blot analysis of TF1-GFP and TF1-PRL-3 cells. GAPDH, loading control. Flow cytometry analysis of propidium iodide-stained TF1-GFP and TF1-PRL-3 after 48 h culture in the absence of cytokines. Note the difference in sub-G1 peak/population, reflective of apoptotic cells. Representative data from three independent experiments are shown. Left panel: flow cytometry analysis of annexin-V- and 7-AAD-stained TF1-GFP and TF1-PRL-3 after 48 h culture in the absence of cytokines. The percentage in the upper left quadrant indicates the fraction of annexin-V-positive apoptotic cells in the entire cell population analysed. Right panel: quantitation of annexin-V-positive apoptotic population in three independent experiments. Statistical differences between two groups were determined using Student's t -test (mean ± SD, n = 3, *** p = 0.0012).

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: MTS Assay, Western Blot, Flow Cytometry, Staining, Quantitation Assay

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: The growth of PRL-3-depleted MOLM-14 and MV4-11 FLT3-ITD-positive AML cells was analysed by MTS assay and flow cytometry. (a) Knock-down of PRL-3 decreased cell number in FLT3-ITD positive MOLM-14 cells (mean ± SD, n = 3). (b) Depletion of PRL-3 accumulated cells in G1 phase in MOLM-14 cells. (a) Knock-down of PRL-3 decreased cell number in FLT3-ITD positive MV4-11 cells (mean ± SD, n = 3). (b) Depletion of PRL-3 accumulated cells in G1 phase in MV4-11 cells. Representative data (right panel) from three independent experiments are shown.

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: MTS Assay, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: (a, b) Results of immunotherapy on liver and spleen sizes in a mouse model of AML. (a) Representative images of livers and spleens harvested from normal nude mice (upper left panel) or nude mice 12–14 days after i.v. injection of TF1-ITD cells together with bi-weekly i.v. administration of control IgG (upper right panel), PRL-3 mAb (lower left panel) or FLT3 mAb (lower right panel). (b) Quantitation of liver and spleen weights of mice as described in (a). Statistical differences between data groups were determined using Student's t -test from three independent experiments. * p < 0.001; ** p = 0.00121. (a, b) Results of immunotherapy and PRL-3 knock-down on leukaemic infiltration in mouse bone marrow (BM) cells in a mouse model of AML. (a) BM cells from nude mice 12–14 days after i.v. injection of (I) TF1, TF1-ITD cells together with bi-weekly i.v. administration of (II) control IgG or (III) PRL-3 mAb (lower left panel) or (IV) TF1-ITD cells depleted of endogenous PRL-3 were analysed using flow cytometry analysis using the human-specific marker CD45+ to distinguish TF1 human-derived AML cells. Percentages indicate proportion of CD45+ cells in the BM population analysed. (b) Quantitation of CD45+ engrafted cells as described in (a). Statistical differences between two groups were determined using Student's t -test (mean ± SD, n = 5, * p < 0.001). Kaplan–Meier survival analysis of PRL-3 mAb-treated ( n = 7) or control IgG-treated ( n = 7) mice in the TF1-ITD leukaemia mouse model ( p < 0.001).

Article Snippet: To generate PRL-3 knock-down cell lines, pRS-PRL-3-shRNA (OriGene Technologies) was transfected into TF1-ITD cells.

Techniques: Injection, Quantitation Assay, Flow Cytometry, Marker, Derivative Assay

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2

doi: 10.3389/fcimb.2015.00082

Figure Lengend Snippet: Determine the binding of the recombinant proteins to LN and FN by Far-Western blot . Coomassie G-250-stained gel (A) , Western blot analysis (B) , and Far-Western blot analysis (C,D) of the SS2 recombinant proteins. Recombinant proteins were separated by 12% SDS-PAGE, then transferred to PVDF membrane and incubated with human LN or LN. Bound LN or FN was detected with goat anti-rabbit IgG antibody. The Western blot (B) was probed with his tag monoclonal antibody (Boster).

Article Snippet: His tag monoclonal antibody (Boster; 1:1000 dilution) were added to every plate and wells were then incubated at 37°C for 30 min. After washing, the cultured cells were incubated with goat anti-mouse IgG-FITC (Boster; 1:2000 dilution) at 37°C for 30 min. After a finial wash, the samples were examined using a fluorescence microscope (ZEISS, Germany).

Techniques: Binding Assay, Recombinant, Far Western Blot, Staining, Western Blot, SDS Page, Membrane, Incubation

Journal: Scientific Reports

Article Title: Functional compensation of glutathione S-transferase M1 ( GSTM1 ) null by another GST superfamily member, GSTM2

doi: 10.1038/srep02704

Figure Lengend Snippet: Normalised gene expression profile (mean ± SEM) for GST family members in 15 GSTM1 -null individuals with respect to the 15 age and sex-matched GSTM1 -positive individuals. The significantly high GSTM2 expression (2.4 fold) under normal physiological conditions indicates a compensatory mechanism in the individuals completely lacking the GSTM1 enzyme.

Article Snippet: GFP-tagged plasmid constructs for GSTM1 and GSTM2 were purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Functional compensation of glutathione S-transferase M1 ( GSTM1 ) null by another GST superfamily member, GSTM2

doi: 10.1038/srep02704

Figure Lengend Snippet: Representative figure for (a) β-actin from one GSTM1 positive (Lane 1) and two null individuals (Lanes 2 and 3). (b) A GSTM1 -positive individual showing a specific 26-kDa band for GSTM1, whereas no band is observed for the GSTM1 -null individuals. (c) GSTM2 is present in all three individuals, though with various intensities. (d) A densitometric analysis (mean ± SEM; pixels/ng) of the target proteins in the western blot. A total of 17 GSTM1 null and 16 GSTM1 positive samples were analyzed. All the blots are representative cropped images and every set have been processed simultaenously, under similar conditions. Representative original blots with cropped demarcations (3b & 3c) are provided in supplementary figure 1.

Article Snippet: GFP-tagged plasmid constructs for GSTM1 and GSTM2 were purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Functional compensation of glutathione S-transferase M1 ( GSTM1 ) null by another GST superfamily member, GSTM2

doi: 10.1038/srep02704

Figure Lengend Snippet: Group (a): i) Trypan blue cell viability assay for GSTM1 in HeLa cells. ii) Western blotting analysis of GSTM1 overexpression in transfected (C) and control cells (V). iii) GFP profile in transfected HeLa cells. Group (b): i) Trypan blue cell viability assay of HeLa cells transfected with GSTM2 and treated either with scrambled or GSTM1 -targeted si-RNA (V-, empty vector without GSH-SF treatment, V+, empty vector with GSH-SF treatment, C-, plasmid construct with either GSTM1 (in figure a) or GSTM2 (in figure b) without GSH-SF treatment, C+, plasmid construct with GSH-SF treatment). ii) Western blotting analysis of GSTM1 in the siRNA-treated HeLa cells. iii) Western blot analysis of GSTM2 in siRNA-treated HeLa cells. iv) GFP profile of transfected HeLa cells after siRNA treatment. All the blots are representative cropped images and every set have been processed under similar conditions as detailed in the methods section.

Article Snippet: GFP-tagged plasmid constructs for GSTM1 and GSTM2 were purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Viability Assay, Western Blot, Over Expression, Transfection, Plasmid Preparation, Construct