genomic dna fragments encoding prkg2 exon iii  (Millipore)

 
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    Structured Review

    Millipore genomic dna fragments encoding prkg2 exon iii
    NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for <t>prkg2</t> alleles flanked by LoxP sites (“floxed” PRKG2 f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μM NO-Cbi or vehicle for 10 min. Akt and GSK-3β phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3β(pSer 9 ), with total GSK-3β serving as a loading control; densitometric quantification is shown on the right, with relative amounts of pAkt and pGSK-3β found in vehicle-treated, control virus-infected cells assigned a value of 1. (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into <t>DNA</t> for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μM NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family-1a (Wnt1a), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (βCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tuba1) mRNA was measured by qRT-PCR and normalized to 18S rRNA; normalized mRNA in untreated cells was assigned a value of 1 . (F) POBs cultured in 10 % FBS were treated with 10 μM NO-Cbi for the indicated times, and Lrp5 protein (open circles) and mRNA (filled squares) were assessed by Western blotting (a representative blot is shown) and qRT-PCR, respectively. Panels A–F show means ± SEM of 3–5 independent experiments in osteoblasts isolated from PRKG2 f/f ). *p

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    1) Product Images from "A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE"

    Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    doi: 10.1002/jbmr.2909

    NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites (“floxed” PRKG2 f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μM NO-Cbi or vehicle for 10 min. Akt and GSK-3β phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3β(pSer 9 ), with total GSK-3β serving as a loading control; densitometric quantification is shown on the right, with relative amounts of pAkt and pGSK-3β found in vehicle-treated, control virus-infected cells assigned a value of 1. (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into DNA for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μM NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family-1a (Wnt1a), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (βCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tuba1) mRNA was measured by qRT-PCR and normalized to 18S rRNA; normalized mRNA in untreated cells was assigned a value of 1 . (F) POBs cultured in 10 % FBS were treated with 10 μM NO-Cbi for the indicated times, and Lrp5 protein (open circles) and mRNA (filled squares) were assessed by Western blotting (a representative blot is shown) and qRT-PCR, respectively. Panels A–F show means ± SEM of 3–5 independent experiments in osteoblasts isolated from PRKG2 f/f ). *p
    Figure Legend Snippet: NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites (“floxed” PRKG2 f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μM NO-Cbi or vehicle for 10 min. Akt and GSK-3β phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3β(pSer 9 ), with total GSK-3β serving as a loading control; densitometric quantification is shown on the right, with relative amounts of pAkt and pGSK-3β found in vehicle-treated, control virus-infected cells assigned a value of 1. (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into DNA for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μM NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family-1a (Wnt1a), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (βCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tuba1) mRNA was measured by qRT-PCR and normalized to 18S rRNA; normalized mRNA in untreated cells was assigned a value of 1 . (F) POBs cultured in 10 % FBS were treated with 10 μM NO-Cbi for the indicated times, and Lrp5 protein (open circles) and mRNA (filled squares) were assessed by Western blotting (a representative blot is shown) and qRT-PCR, respectively. Panels A–F show means ± SEM of 3–5 independent experiments in osteoblasts isolated from PRKG2 f/f ). *p

    Techniques Used: Isolation, Mouse Assay, Infection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Cell Culture, ALP Assay

    NO-Cbi inhibits osteoclast differentiation (A,B) Murine bone marrow mono-nuclear cells were cultured in the presence of M-CSF; after 3 d, RANKL was added, with or without NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8. (C) Cells were cultured as in A, but some cultures received 10 μM NO-Cbi, 5 μM DETA-NONOate (Deta-NO, which releases two moles of NO per mole of drug), or 100 μM 8-pCPT-cGMP together with RANKL. (D) Expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. (E–G) Bone marrow mononuclear cells isolated from PRKG2 f/f mice were cultured with M-CSF and RANKL as described in panel A, but 24 h after plating, cells were infected with adenovirus expressing either green fluorescent protein (GFP, control) or CRE. Relative amounts of prkg2 mRNA were determined by qRT-PCR (E), and TRAP-positive cells were counted on day 8 (F,G). Panels B–F show means ± SEM of at least three independent experiments; *p
    Figure Legend Snippet: NO-Cbi inhibits osteoclast differentiation (A,B) Murine bone marrow mono-nuclear cells were cultured in the presence of M-CSF; after 3 d, RANKL was added, with or without NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8. (C) Cells were cultured as in A, but some cultures received 10 μM NO-Cbi, 5 μM DETA-NONOate (Deta-NO, which releases two moles of NO per mole of drug), or 100 μM 8-pCPT-cGMP together with RANKL. (D) Expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. (E–G) Bone marrow mononuclear cells isolated from PRKG2 f/f mice were cultured with M-CSF and RANKL as described in panel A, but 24 h after plating, cells were infected with adenovirus expressing either green fluorescent protein (GFP, control) or CRE. Relative amounts of prkg2 mRNA were determined by qRT-PCR (E), and TRAP-positive cells were counted on day 8 (F,G). Panels B–F show means ± SEM of at least three independent experiments; *p

    Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Infection

    NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation, and survival in POBs (A) POBs isolated from intact C57Bl6 mice were incubated in medium with 0.1% FBS (3 × 10 5 cells/ml) for 2 h prior to receiving vehicle or 10 μM NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NO x ) were measured in the medium by the Griess reaction (NO x present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min. The intracellular cGMP concentration was measured by ELISA (B), and VASP phosphorylation was analyzed by Western blot using a phospho-Ser 259 -specific antibody, with bar graph showing densitometric quantification of pVASP normalized to β-actin (C). (D,E) Serum-deprived POBs were treated with vehicle or 10 μM NO-Cbi for 10 min and ERK ( D ) and Akt ( E ) activation were assessed by blotting with phospho-specific antibodies. Densitometric quantification of pErk and pAkt normalized to total Erk and Akt, respectively, is shown in the bar graphs. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μM NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs were cultured in medium with 0.1% FBS for 18 h, and treated with 10 μM NO-Cbi or vehicle for 1 h; they were transferred to fresh medium containing 3 H-thymidine for 24 h, and thymidine incorporation into DNA was measured. ( H,I ) Confluent POBs were differentiated in ascorbate-containing medium for 14 d, with some cells receiving 10 μM NO-Cbi (filled bars) or vehicle (open bars) during the last 24 h. Expression of osteocalcin (OCN), osteopontin (Spp1), collagen 1-A1 (Col1a1), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tuba1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. Panels A–I show means ± SEM of at least three independent experiments; *p
    Figure Legend Snippet: NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation, and survival in POBs (A) POBs isolated from intact C57Bl6 mice were incubated in medium with 0.1% FBS (3 × 10 5 cells/ml) for 2 h prior to receiving vehicle or 10 μM NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NO x ) were measured in the medium by the Griess reaction (NO x present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min. The intracellular cGMP concentration was measured by ELISA (B), and VASP phosphorylation was analyzed by Western blot using a phospho-Ser 259 -specific antibody, with bar graph showing densitometric quantification of pVASP normalized to β-actin (C). (D,E) Serum-deprived POBs were treated with vehicle or 10 μM NO-Cbi for 10 min and ERK ( D ) and Akt ( E ) activation were assessed by blotting with phospho-specific antibodies. Densitometric quantification of pErk and pAkt normalized to total Erk and Akt, respectively, is shown in the bar graphs. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μM NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs were cultured in medium with 0.1% FBS for 18 h, and treated with 10 μM NO-Cbi or vehicle for 1 h; they were transferred to fresh medium containing 3 H-thymidine for 24 h, and thymidine incorporation into DNA was measured. ( H,I ) Confluent POBs were differentiated in ascorbate-containing medium for 14 d, with some cells receiving 10 μM NO-Cbi (filled bars) or vehicle (open bars) during the last 24 h. Expression of osteocalcin (OCN), osteopontin (Spp1), collagen 1-A1 (Col1a1), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tuba1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. Panels A–I show means ± SEM of at least three independent experiments; *p

    Techniques Used: Expressing, Isolation, Mouse Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Immunofluorescence, Staining, Cell Culture, ALP Assay, Quantitative RT-PCR

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    Western Blot:

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    Amplification:

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    Clone Assay:

    Article Title: Transcription Elongation Factor GreA Plays a Key Role in Cellular Invasion and Virulence of Francisella tularensis subsp. novicida
    Article Snippet: .. Antibody generation and western blotting Briefly, the greA open reading frame was amplified from the F. novicida U112 genomic DNA with the primer pair Pr0023/Pr0024 (Table ), and cloned into the plasmid pET-32a (Novagen, Madison, WI, USA). .. Escherichia coli BL21 (DE3) was transformed with the resulting plasmid to express the protein, which was purified with Ni–Agarose (Solarbio, Beijing, China).

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    Plasmid Preparation:

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    Article Snippet: .. Antibody generation and western blotting Briefly, the greA open reading frame was amplified from the F. novicida U112 genomic DNA with the primer pair Pr0023/Pr0024 (Table ), and cloned into the plasmid pET-32a (Novagen, Madison, WI, USA). .. Escherichia coli BL21 (DE3) was transformed with the resulting plasmid to express the protein, which was purified with Ni–Agarose (Solarbio, Beijing, China).

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    Positron Emission Tomography:

    Article Title: Transcription Elongation Factor GreA Plays a Key Role in Cellular Invasion and Virulence of Francisella tularensis subsp. novicida
    Article Snippet: .. Antibody generation and western blotting Briefly, the greA open reading frame was amplified from the F. novicida U112 genomic DNA with the primer pair Pr0023/Pr0024 (Table ), and cloned into the plasmid pET-32a (Novagen, Madison, WI, USA). .. Escherichia coli BL21 (DE3) was transformed with the resulting plasmid to express the protein, which was purified with Ni–Agarose (Solarbio, Beijing, China).

    Polymerase Chain Reaction:

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    Article Title: pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion
    Article Snippet: 100 µl HBMECs was added to 100 µl of the pneumococcal suspension in PBS (107 CFU/ml), and the mixture was incubated at 4°C with gentle agitation for 1 h. After cold centrifugation, the supernatant was removed and the bacterial pellet was resuspended in LDS sample buffer 1X LDS-sample buffer (Thermo Fisher Scientific and Invitrogen) and boiled at 95°C for 5 min. Lysates were loaded onto a 10% NuPage Novex Bis-Tris Gel (Thermo Fisher Scientific and Invitrogen), and electroblotting was performed using the Trans-Blot Turbo Transfer System. .. Expression and purification of RrgA and PspA Truncated rrgA encoding amino acids 39–868 was amplified by PCR from S. pneumoniae TIGR4 genomic DNA and ligated into pACYCDuet-1 vector (Novagen) to generate N-terminally 6x-His tagged RrgA39–868 . .. In brief, T7 express competent Escherichia coli cells (New England Biolabs, Inc.) expressing 6xHis- RrgA39–868 were grown overnight at 16°C after induction of protein expression with isopropyl-β-d -thiogalactopyranoside at 1 mM final concentration.

    Article Title: Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex
    Article Snippet: .. Sequencing of genomic PCR products PCR was performed on human, chimpanzee and bonobo genomic DNA using the REDTaq DNA polymerase (Sigma). .. Identical cycling and temperature conditions as used for the genomic qPCR described above (annealing at 60°C, 30 cycles) were applied.

    Article Title: PA0833 Is an OmpA C-Like Protein That Confers Protection Against Pseudomonas aeruginosa Infection
    Article Snippet: Two constructs were made to produce truncated PA0833 proteins comprising residues 26–237 and 86–237. .. In brief, the coding sequences of the two constructs were amplified by PCR from P. aeruginosa strain PAO1 genomic DNA and cloned into the pGEX6p-2 vector (Novagen, Italy) via the Bam H1/ Xho I restriction sites. .. The resulting proteins harbor an N-terminal glutathione S-transferases (GST) tag to facilitate the purification of the proteins.

    Chromatin Immunoprecipitation:

    Article Title: Transcriptional control and exploitation of an immune‐responsive family of plant retrotransposons
    Article Snippet: The same amount of the corresponding non‐digested DNA was used for qPCR as a control and to normalize the data. .. Pyrosequencing ATCOPIA93 DNA (ChIP‐DNA, cDNA, or gDNA as a control) was amplified with a biotinylated (forward) primer in the same region where RNA levels were analyzed and containing a SNP between EVD and ATR ; the biotinylated PCR product (40 μl reaction) was pulled down with streptavidin beads (sigma GE17‐5113‐01) and the sense biotinylated strand sequenced with a Pyromark Q24 (Qiagen) on the sequencing mode. ..

    Sequencing:

    Article Title: Transcriptional control and exploitation of an immune‐responsive family of plant retrotransposons
    Article Snippet: The same amount of the corresponding non‐digested DNA was used for qPCR as a control and to normalize the data. .. Pyrosequencing ATCOPIA93 DNA (ChIP‐DNA, cDNA, or gDNA as a control) was amplified with a biotinylated (forward) primer in the same region where RNA levels were analyzed and containing a SNP between EVD and ATR ; the biotinylated PCR product (40 μl reaction) was pulled down with streptavidin beads (sigma GE17‐5113‐01) and the sense biotinylated strand sequenced with a Pyromark Q24 (Qiagen) on the sequencing mode. ..

    Article Title: Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex
    Article Snippet: .. Sequencing of genomic PCR products PCR was performed on human, chimpanzee and bonobo genomic DNA using the REDTaq DNA polymerase (Sigma). .. Identical cycling and temperature conditions as used for the genomic qPCR described above (annealing at 60°C, 30 cycles) were applied.

    Expressing:

    Article Title: pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion
    Article Snippet: 100 µl HBMECs was added to 100 µl of the pneumococcal suspension in PBS (107 CFU/ml), and the mixture was incubated at 4°C with gentle agitation for 1 h. After cold centrifugation, the supernatant was removed and the bacterial pellet was resuspended in LDS sample buffer 1X LDS-sample buffer (Thermo Fisher Scientific and Invitrogen) and boiled at 95°C for 5 min. Lysates were loaded onto a 10% NuPage Novex Bis-Tris Gel (Thermo Fisher Scientific and Invitrogen), and electroblotting was performed using the Trans-Blot Turbo Transfer System. .. Expression and purification of RrgA and PspA Truncated rrgA encoding amino acids 39–868 was amplified by PCR from S. pneumoniae TIGR4 genomic DNA and ligated into pACYCDuet-1 vector (Novagen) to generate N-terminally 6x-His tagged RrgA39–868 . .. In brief, T7 express competent Escherichia coli cells (New England Biolabs, Inc.) expressing 6xHis- RrgA39–868 were grown overnight at 16°C after induction of protein expression with isopropyl-β-d -thiogalactopyranoside at 1 mM final concentration.

    Purification:

    Article Title: pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion
    Article Snippet: 100 µl HBMECs was added to 100 µl of the pneumococcal suspension in PBS (107 CFU/ml), and the mixture was incubated at 4°C with gentle agitation for 1 h. After cold centrifugation, the supernatant was removed and the bacterial pellet was resuspended in LDS sample buffer 1X LDS-sample buffer (Thermo Fisher Scientific and Invitrogen) and boiled at 95°C for 5 min. Lysates were loaded onto a 10% NuPage Novex Bis-Tris Gel (Thermo Fisher Scientific and Invitrogen), and electroblotting was performed using the Trans-Blot Turbo Transfer System. .. Expression and purification of RrgA and PspA Truncated rrgA encoding amino acids 39–868 was amplified by PCR from S. pneumoniae TIGR4 genomic DNA and ligated into pACYCDuet-1 vector (Novagen) to generate N-terminally 6x-His tagged RrgA39–868 . .. In brief, T7 express competent Escherichia coli cells (New England Biolabs, Inc.) expressing 6xHis- RrgA39–868 were grown overnight at 16°C after induction of protein expression with isopropyl-β-d -thiogalactopyranoside at 1 mM final concentration.

    Construct:

    Article Title: PA0833 Is an OmpA C-Like Protein That Confers Protection Against Pseudomonas aeruginosa Infection
    Article Snippet: Two constructs were made to produce truncated PA0833 proteins comprising residues 26–237 and 86–237. .. In brief, the coding sequences of the two constructs were amplified by PCR from P. aeruginosa strain PAO1 genomic DNA and cloned into the pGEX6p-2 vector (Novagen, Italy) via the Bam H1/ Xho I restriction sites. .. The resulting proteins harbor an N-terminal glutathione S-transferases (GST) tag to facilitate the purification of the proteins.

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    Millipore genomic dna fragments encoding prkg2 exon iii
    NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for <t>prkg2</t> alleles flanked by LoxP sites (“floxed” PRKG2 f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μM NO-Cbi or vehicle for 10 min. Akt and GSK-3β phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3β(pSer 9 ), with total GSK-3β serving as a loading control; densitometric quantification is shown on the right, with relative amounts of pAkt and pGSK-3β found in vehicle-treated, control virus-infected cells assigned a value of 1. (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into <t>DNA</t> for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μM NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family-1a (Wnt1a), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (βCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tuba1) mRNA was measured by qRT-PCR and normalized to 18S rRNA; normalized mRNA in untreated cells was assigned a value of 1 . (F) POBs cultured in 10 % FBS were treated with 10 μM NO-Cbi for the indicated times, and Lrp5 protein (open circles) and mRNA (filled squares) were assessed by Western blotting (a representative blot is shown) and qRT-PCR, respectively. Panels A–F show means ± SEM of 3–5 independent experiments in osteoblasts isolated from PRKG2 f/f ). *p
    Genomic Dna Fragments Encoding Prkg2 Exon Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites (“floxed” PRKG2 f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μM NO-Cbi or vehicle for 10 min. Akt and GSK-3β phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3β(pSer 9 ), with total GSK-3β serving as a loading control; densitometric quantification is shown on the right, with relative amounts of pAkt and pGSK-3β found in vehicle-treated, control virus-infected cells assigned a value of 1. (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into DNA for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μM NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family-1a (Wnt1a), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (βCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tuba1) mRNA was measured by qRT-PCR and normalized to 18S rRNA; normalized mRNA in untreated cells was assigned a value of 1 . (F) POBs cultured in 10 % FBS were treated with 10 μM NO-Cbi for the indicated times, and Lrp5 protein (open circles) and mRNA (filled squares) were assessed by Western blotting (a representative blot is shown) and qRT-PCR, respectively. Panels A–F show means ± SEM of 3–5 independent experiments in osteoblasts isolated from PRKG2 f/f ). *p

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE

    doi: 10.1002/jbmr.2909

    Figure Lengend Snippet: NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II (A) POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites (“floxed” PRKG2 f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μM NO-Cbi or vehicle for 10 min. Akt and GSK-3β phosphorylation were assessed using antibodies specific for Akt(pSer 473 ) and GSK-3β(pSer 9 ), with total GSK-3β serving as a loading control; densitometric quantification is shown on the right, with relative amounts of pAkt and pGSK-3β found in vehicle-treated, control virus-infected cells assigned a value of 1. (C) PRKG2 f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring 3 H-thymidine incorporation into DNA for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μM NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family-1a (Wnt1a), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (βCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tuba1) mRNA was measured by qRT-PCR and normalized to 18S rRNA; normalized mRNA in untreated cells was assigned a value of 1 . (F) POBs cultured in 10 % FBS were treated with 10 μM NO-Cbi for the indicated times, and Lrp5 protein (open circles) and mRNA (filled squares) were assessed by Western blotting (a representative blot is shown) and qRT-PCR, respectively. Panels A–F show means ± SEM of 3–5 independent experiments in osteoblasts isolated from PRKG2 f/f ). *p

    Article Snippet: To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).

    Techniques: Isolation, Mouse Assay, Infection, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Cell Culture, ALP Assay

    NO-Cbi inhibits osteoclast differentiation (A,B) Murine bone marrow mono-nuclear cells were cultured in the presence of M-CSF; after 3 d, RANKL was added, with or without NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8. (C) Cells were cultured as in A, but some cultures received 10 μM NO-Cbi, 5 μM DETA-NONOate (Deta-NO, which releases two moles of NO per mole of drug), or 100 μM 8-pCPT-cGMP together with RANKL. (D) Expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. (E–G) Bone marrow mononuclear cells isolated from PRKG2 f/f mice were cultured with M-CSF and RANKL as described in panel A, but 24 h after plating, cells were infected with adenovirus expressing either green fluorescent protein (GFP, control) or CRE. Relative amounts of prkg2 mRNA were determined by qRT-PCR (E), and TRAP-positive cells were counted on day 8 (F,G). Panels B–F show means ± SEM of at least three independent experiments; *p

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE

    doi: 10.1002/jbmr.2909

    Figure Lengend Snippet: NO-Cbi inhibits osteoclast differentiation (A,B) Murine bone marrow mono-nuclear cells were cultured in the presence of M-CSF; after 3 d, RANKL was added, with or without NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8. (C) Cells were cultured as in A, but some cultures received 10 μM NO-Cbi, 5 μM DETA-NONOate (Deta-NO, which releases two moles of NO per mole of drug), or 100 μM 8-pCPT-cGMP together with RANKL. (D) Expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. (E–G) Bone marrow mononuclear cells isolated from PRKG2 f/f mice were cultured with M-CSF and RANKL as described in panel A, but 24 h after plating, cells were infected with adenovirus expressing either green fluorescent protein (GFP, control) or CRE. Relative amounts of prkg2 mRNA were determined by qRT-PCR (E), and TRAP-positive cells were counted on day 8 (F,G). Panels B–F show means ± SEM of at least three independent experiments; *p

    Article Snippet: To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Isolation, Mouse Assay, Infection

    NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation, and survival in POBs (A) POBs isolated from intact C57Bl6 mice were incubated in medium with 0.1% FBS (3 × 10 5 cells/ml) for 2 h prior to receiving vehicle or 10 μM NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NO x ) were measured in the medium by the Griess reaction (NO x present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min. The intracellular cGMP concentration was measured by ELISA (B), and VASP phosphorylation was analyzed by Western blot using a phospho-Ser 259 -specific antibody, with bar graph showing densitometric quantification of pVASP normalized to β-actin (C). (D,E) Serum-deprived POBs were treated with vehicle or 10 μM NO-Cbi for 10 min and ERK ( D ) and Akt ( E ) activation were assessed by blotting with phospho-specific antibodies. Densitometric quantification of pErk and pAkt normalized to total Erk and Akt, respectively, is shown in the bar graphs. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μM NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs were cultured in medium with 0.1% FBS for 18 h, and treated with 10 μM NO-Cbi or vehicle for 1 h; they were transferred to fresh medium containing 3 H-thymidine for 24 h, and thymidine incorporation into DNA was measured. ( H,I ) Confluent POBs were differentiated in ascorbate-containing medium for 14 d, with some cells receiving 10 μM NO-Cbi (filled bars) or vehicle (open bars) during the last 24 h. Expression of osteocalcin (OCN), osteopontin (Spp1), collagen 1-A1 (Col1a1), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tuba1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. Panels A–I show means ± SEM of at least three independent experiments; *p

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE

    doi: 10.1002/jbmr.2909

    Figure Lengend Snippet: NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation, and survival in POBs (A) POBs isolated from intact C57Bl6 mice were incubated in medium with 0.1% FBS (3 × 10 5 cells/ml) for 2 h prior to receiving vehicle or 10 μM NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NO x ) were measured in the medium by the Griess reaction (NO x present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min. The intracellular cGMP concentration was measured by ELISA (B), and VASP phosphorylation was analyzed by Western blot using a phospho-Ser 259 -specific antibody, with bar graph showing densitometric quantification of pVASP normalized to β-actin (C). (D,E) Serum-deprived POBs were treated with vehicle or 10 μM NO-Cbi for 10 min and ERK ( D ) and Akt ( E ) activation were assessed by blotting with phospho-specific antibodies. Densitometric quantification of pErk and pAkt normalized to total Erk and Akt, respectively, is shown in the bar graphs. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μM NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs were cultured in medium with 0.1% FBS for 18 h, and treated with 10 μM NO-Cbi or vehicle for 1 h; they were transferred to fresh medium containing 3 H-thymidine for 24 h, and thymidine incorporation into DNA was measured. ( H,I ) Confluent POBs were differentiated in ascorbate-containing medium for 14 d, with some cells receiving 10 μM NO-Cbi (filled bars) or vehicle (open bars) during the last 24 h. Expression of osteocalcin (OCN), osteopontin (Spp1), collagen 1-A1 (Col1a1), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tuba1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. Panels A–I show means ± SEM of at least three independent experiments; *p

    Article Snippet: To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).

    Techniques: Expressing, Isolation, Mouse Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Immunofluorescence, Staining, Cell Culture, ALP Assay, Quantitative RT-PCR