polymerase chain reaction pcr mixture  (Roche)

Journal: Journal of Signal Transduction

Article Title: A Novel Interaction between Pyk2 and MAP4K4 Is Integrated with Glioma Cell Migration

doi: 10.1155/2013/956580

Figure Lengend Snippet: Knockdown of MAP4K4 expression inhibits glioma cell migration and invasion and blocks Pyk2 mediated stimulation of migration. (a) SF767 cells were transiently transfected with shRNA 1M4K4i and shRNA 2M4K4i or cotransfected with shRNAs 1M4K41i and 2M4K4i. Cell lysates were blotted with indicated antibodies. (b) SF767 cells were stably transduced with lentivirus expressing 2M4K4i shRNA. Stably transduced cells (solid histogram) were enriched by flow cytometry. Open histogram: control SF767 parental cells. Inset: whole cell lysates of control SF767 cells or SF767 cells stably transduced with MAP4K4 shRNA (MAP4K4i) were immunoblotted with the indicated antibodies. (c) The migration rate of control SF767 cells, SF767 cells stably transduced with a shRNA targeting MAP4K4 (MAP4K4i), and MAP4K4i cells transfected with Pyk2 was assayed with a radial migration assay on 10 μ g/mL laminin substrate. * P

Article Snippet: The coding sequence for full length MAP4K4 was isolated by RT-PCR of total RNA isolated from the SF767 glioma cell line using the Titan RT-PCR kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions.

Techniques: Expressing, Migration, Transfection, shRNA, Stable Transfection, Transduction, Flow Cytometry, Cytometry

Journal: Journal of Signal Transduction

Article Title: A Novel Interaction between Pyk2 and MAP4K4 Is Integrated with Glioma Cell Migration

doi: 10.1155/2013/956580

Figure Lengend Snippet: Schematic of MAP4K4 and interacting two-hybrid clones. (a) Structure of full length MAP4K4 indicating the N-terminal kinase domain, the C-terminal citron homology domain (CNH), and the location of alternatively spliced modules M1–M9. (b) Structure of the three overlapping clones isolated in the yeast two-hybrid genetic screen which interact with the Pyk2 FERM domain. (c) Structure of the final MAP4K4 clone assembled from SF767 glioma cell isoforms.

Article Snippet: The coding sequence for full length MAP4K4 was isolated by RT-PCR of total RNA isolated from the SF767 glioma cell line using the Titan RT-PCR kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions.

Techniques: Clone Assay, Isolation

Journal: Journal of Signal Transduction

Article Title: A Novel Interaction between Pyk2 and MAP4K4 Is Integrated with Glioma Cell Migration

doi: 10.1155/2013/956580

Figure Lengend Snippet: Pyk2 coimmunoprecipitates with MAP4K4. (a) Cells cotransfected with FLAG-epitope-tagged Pyk2 FERM and HA-epitope-tagged MAP4K4(143) were lysed and immunoprecipitated with anti-FLAG antibody or normal mouse IgG. Immunoprecipitates were immunoblotted (IB) with anti-FLAG or anti-HA. WCL: whole cell lysate. (b) Cells were cotransfected with FLAG-epitope-tagged MAP4K4(143) and either HA-tagged Pyk2 or HA-tagged FAK, lysed, and immunoprecipitated with mouse IgG or anti-FLAG antibody. Immunoprecipitates or WCL were immunoblotted as indicated. (c) Cells cotransfected with FLAG-tagged Pyk2 and either HA-tagged MAP4K4(143) or HA-tagged full length (FL) MAP4K4 were lysed and immunoprecipitated with rabbit IgG or anti-HA antibody. Immunoprecipitates or whole cell lysate (WCL) was immunoblotted with the indicated antibodies. (d) SF767 glioma cells were lysed and immunoprecipitated with anti-MAP4K4 antibody. Immunoprecipitates or whole cell lysate (WCL) was immunoblotted with the indicated antibodies.

Article Snippet: The coding sequence for full length MAP4K4 was isolated by RT-PCR of total RNA isolated from the SF767 glioma cell line using the Titan RT-PCR kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions.

Techniques: FLAG-tag, Immunoprecipitation

Journal: Journal of Signal Transduction

Article Title: A Novel Interaction between Pyk2 and MAP4K4 Is Integrated with Glioma Cell Migration

doi: 10.1155/2013/956580

Figure Lengend Snippet: Increased expression of MAP4K4 stimulates migration but is blocked by knockdown of Pyk2 expression. (a) Wild-type SF767 glioma cells (CTL) or SF767 cells stably transduced with a shRNA targeting Pyk2 (Pyk2i) were transfected with vector or HA-epitope-tagged MAP4K4. Whole cell lysates were blotted with the indicated antibodies. Lysates were blotted with actin as a loading control. (b) SF767 control cells or SF767 cells with shRNA mediated knockdown of Pyk2 (Pyk2i) were transfected with MAP4K4, and the migration rate on 10 μ g/mL laminin was assessed over 24 hr using a radial migration assay. † P = 0.0016 relative to control. * P

Article Snippet: The coding sequence for full length MAP4K4 was isolated by RT-PCR of total RNA isolated from the SF767 glioma cell line using the Titan RT-PCR kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions.

Techniques: Expressing, Migration, CTL Assay, Stable Transfection, Transduction, shRNA, Transfection, Plasmid Preparation

Journal: BMC Microbiology

Article Title: Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture

doi: 10.1186/1471-2180-2-12

Figure Lengend Snippet: Detection of HSV DNA from clinical samples by LightCycler PCR.

Article Snippet: The LC-PCR master mix contained the following: 1× FastStart Taq DNA polymerase reaction buffer (Roche Molecular Diagnostics) which included a dNTP mix (containing dUTP instead of dTTP), 3 mM MgCl2 , 0.5 μM of each primer, 0.2 μM HSV-2 FLU and 0.4 μM HSV-2 LCR.

Techniques: Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines

doi: 10.1371/journal.pone.0125622

Figure Lengend Snippet: Detection of RT activity in cell lines infected with MLV of contaminated cell lines by PERT assay. Cell culture supernatants of the infected cell lines NCI-H82 and VERO-B4 were harvested after 45 and 44 days after infection, respectively. MS2 phage RNA was reverse transcribed with an MS2-specific primer and the MLV derived RT of the samples in the presence of MnCl 2 . The cDNA was then amplified with a second MS2-specific primer applying PCR. The signals of the cell lines infected with LCL-HO supernatant may potentially be ascribed to the activity of SMRV derived RT. dpi: days post infection.

Article Snippet: Primer template annealing One μl of PERT-RT1R primer at 10 mM was mixed with 0.4 μl bacteriophage MS2 RNA at 0.8 μg/μl (Roche Diagnostics, Mannheim, Germany) per reaction, denatured at 95°C for 5 min and subsequently incubated for 30 min at 37°C for annealing of the primer to the template.

Techniques: Activity Assay, Infection, Cell Culture, Derivative Assay, Amplification, Polymerase Chain Reaction

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: MOR agonists increase MMP-9 expression in dissociated neurons of DRG cultures . (A) Triple staining of MMP-9, NeuN (neuronal marker), and DAPI (nuclei marker for all cells). Note that MMP-9 is only expressed in neurons (arrows). Scale, 100 μm. (B) Double staining showing MMP-9 and NeuN expression in dissociated DRG neurons in control cultures and cultures treated with morphine (10 μM, 2 h). Left panels show all neurons (NeuN+) in the field. Right panels show high magnification images of middle panels. Scales, 100 μm (middle) and 25 μm (right). (C) Intensity of MMP-9-positive neurons in DRG cultures after treatment of morphine (1, 10, and 50 μM, 2 h), DAMGO and remifentanil (1 and 10 μM, 2 h), together with the opioid receptor antagonist naloxone and MOR antagonist CTAT (10 and 50 μM). Note that morphine-induced MMP-9 increase is inhibited by naloxone and CTAP. * P

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques: Expressing, Staining, Marker, Double Staining

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: Up-regulation of MMP-9 expression in DRGs but not spinal cords after intrathecal morphine or remifentanil and potentiation of intrathecal morphine analgesia in Mmp9 deficient mice . (A-D) Western blotting showing MMP-9 expression in DRGs (A, B) and spinal cord dorsal horns (C, D) 2 h after intrathecal injection of morphine (10 nmol) or remifentanil (1 nmol). B and D are quantification of the MMP-9 bands in DRGs (B) and spinal cords (D). * P

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques: Expressing, Mouse Assay, Western Blot, Injection

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: Intrathecal administration of MMP-9 siRNA reduces MMP-9 expression in DRGs and enhances morphine analgesia . (A-D) Western blotting showing MMP-9 expression in DRGs (A, B) and spinal cord dorsal horns (C, D) after intrathecal injections of MMP-9 siRNA or control siRNA (3 μg, 24 and 2 h before the s.c. morphine injection. B and D are quantification of the MMP-9 bands in DRGs (B) and spinal cords (D). * P

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques: Expressing, Western Blot, Injection

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: MMP-9 deficiency does not alter opioid-induced hyperalgesia after subcutaneous morphine . (A, B) Western blot gel (A) and band density (B) showing MMP-9 expression in lumbar DRGs 24 h after morphine (MORPH) injection. * P > 0.05, compared to naive control (CTRL), Student's t-test, n = 4 mice. (C, D) Subcutaneous morphine (5 mg/kg) induces comparable heat hyperalgesia in both wild-type and Mmp9 -KO mice (C) and also in wild-type mice pretreated with intrathecal MMP-9 inhibitor-I (0.2 μg) or vehicle (10% DMSO). * P

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques: Western Blot, Expressing, Injection, Mouse Assay

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: Subcutaneous morphine induces MMP-9 and TIMP-1 mRNA up-regulation in DRGs . (A, B) RT-PCR showing time course of subcutaneous morphine-induced MMP-9 (A) and TIMP-1 (B) mRNA expression in DRGs. * P

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: Subcutaneous morphine-induced MMP-9 increase is enriched in mu opioid receptor (MOR)-expressing DRG neurons . (A, B) Double staining showing colocalization of MMP-9 and MOR in DRG neurons of saline (A) and morphine (B) treated mice (s.c., 10 mg/kg). Scale, 50 μm. (C, D) Percentage of MMP-9+ (C) and MOR + (D) neurons in DRGs 2 h after saline and morphine injection. (E) Percentage of MMP-9 and MOR double-positive neurons in DRGs 2 h after saline and morphine injection. (F) Percentage of MMP-9-positive neurons expressing MOR and percentage of MOR-positive neurons expressing MMP-9 in DRGs 2 h after saline and morphine injection. S, saline; M, morphine. * P

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques: Expressing, Double Staining, Mouse Assay, Injection

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: Actue morphine treatment does not change the sizes of MMP-9- and MOR-positive neurons in DRGs . (A, B) Size frequency distribution (percentage) of MMP-9-positive neurons (A) and MOR-positive neurons (B) in DRGs 2 h after subcutaneous saline and morphine. 300-500 neurons from 3 animals were plotted in each group. Note that MMP-9 and MOR are mainly expressed in small- and medium-sized neurons.

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques:

Journal: Molecular Pain

Article Title: Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

doi: 10.1186/1744-8069-8-19

Figure Lengend Snippet: Subcutaneous morphine increases MMP-9 expression and activity in DRGs . (A) Western blotting showing the time course of MMP-9 expression in lumbar DRGs after morphine (s.c., 10 mg/kg). (B) Gelatin zymography showing the time course of MMP-9 activity in lumbar DRGs after morphine. Note that MMP-2 activity remains unchanged after morphine injection. (C) Western blotting showing the time course of MMP-9 expression in lumbar spinal cord dorsal horns after morphine (s.c., 10 mg/kg). Note that subcutaneous morphine only induces MMP-9 expression in DRGs but not spinal cords. * P

Article Snippet: Primary DRG cultures and immunocytochemistry DRGs were removed aseptically from 4-week old mice and first incubated with collagenase (1.25 mg/ml, Roche)/dispase-II (2.4 units/ml, Roche) at 37°C for 90 min, then digested with 0.25% trypsin (Cellgro) for 8 min at 37°C, followed by 0.25% trypsin inhibitor (Sigma).

Techniques: Expressing, Activity Assay, Western Blot, Zymography, Injection