PMC:3245220 / 44679-45797 JSONTXT

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T21226","span":{"begin":338,"end":342},"obj":"P17252"},{"id":"T21227","span":{"begin":447,"end":451},"obj":"P17252"},{"id":"T21228","span":{"begin":461,"end":464},"obj":"P21579"},{"id":"T21229","span":{"begin":461,"end":464},"obj":"Q04206"},{"id":"T21230","span":{"begin":821,"end":826},"obj":"P09603"},{"id":"T21231","span":{"begin":823,"end":826},"obj":"P04141"},{"id":"T21232","span":{"begin":954,"end":963},"obj":"P08758"},{"id":"T21233","span":{"begin":1112,"end":1117},"obj":"P09603"},{"id":"T21234","span":{"begin":1114,"end":1117},"obj":"P04141"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

{"project":"bionlp-st-ge-2016-reference","denotations":[{"id":"T21235","span":{"begin":338,"end":348},"obj":"Protein"},{"id":"T21236","span":{"begin":447,"end":451},"obj":"Protein"},{"id":"T21237","span":{"begin":461,"end":464},"obj":"Protein"},{"id":"T21238","span":{"begin":954,"end":963},"obj":"Protein"},{"id":"T21239","span":{"begin":821,"end":826},"obj":"Protein"},{"id":"T21240","span":{"begin":1112,"end":1117},"obj":"Protein"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

{"project":"bionlp-st-ge-2016-reference-tees","denotations":[{"id":"T21292","span":{"begin":338,"end":365},"obj":"Protein"},{"id":"T21293","span":{"begin":447,"end":451},"obj":"Protein"},{"id":"T21294","span":{"begin":455,"end":475},"obj":"Protein"},{"id":"T21295","span":{"begin":821,"end":826},"obj":"Protein"},{"id":"T21296","span":{"begin":915,"end":919},"obj":"Protein"},{"id":"T21297","span":{"begin":1112,"end":1117},"obj":"Protein"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

{"project":"events-check-again","denotations":[{"id":"T21286","span":{"begin":338,"end":348},"obj":"Protein"},{"id":"T21287","span":{"begin":447,"end":451},"obj":"Protein"},{"id":"T21288","span":{"begin":461,"end":464},"obj":"Protein"},{"id":"T21289","span":{"begin":821,"end":826},"obj":"Protein"},{"id":"T21290","span":{"begin":954,"end":963},"obj":"Protein"},{"id":"T21291","span":{"begin":1112,"end":1117},"obj":"Protein"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

{"project":"GO-CC","denotations":[{"id":"T21252","span":{"begin":288,"end":293},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T21253","span":{"begin":564,"end":569},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T21254","span":{"begin":717,"end":722},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T21255","span":{"begin":1015,"end":1020},"obj":"http://purl.obolibrary.org/obo/GO_0005623"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

{"project":"GO-MF","denotations":[{"id":"T21250","span":{"begin":338,"end":342},"obj":"http://purl.obolibrary.org/obo/GO_0004697"},{"id":"T21251","span":{"begin":447,"end":451},"obj":"http://purl.obolibrary.org/obo/GO_0004697"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

{"project":"UBERON-AE","denotations":[{"id":"T20813","span":{"begin":648,"end":653},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T20814","span":{"begin":783,"end":788},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

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Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}

{"project":"test2","denotations":[{"id":"T20807","span":{"begin":338,"end":348},"obj":"Protein"},{"id":"T20808","span":{"begin":447,"end":451},"obj":"Protein"},{"id":"T20809","span":{"begin":461,"end":464},"obj":"Protein"},{"id":"T20810","span":{"begin":821,"end":826},"obj":"Protein"},{"id":"T20811","span":{"begin":954,"end":963},"obj":"Protein"},{"id":"T20812","span":{"begin":1112,"end":1117},"obj":"Protein"}],"text":"Transient Transfection of Primary Human Monocytes\nMonocytes were transfected using the human monocyte Amaxa nucleofector kit (Lonza Walkersville Inc, Walkersville, MD) as previously described [51]. Briefly, monocytes were resuspended in Amaxa Nucleofactor solution at a density of 20×106 cells/ml, and 2 µg of total plasmid DNA 100 nM of PKCα siRNA or control siRNA was added and transfected using program Amaxa Y-01. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. Immediately after transfection, cells were washed with 1 ml of RPMI medium containing 2 mM glutamine and 10% FBS or serum free LGM medium (Lonza Walkersville Inc.) then plated at 4×105 cells/well in 24-well plates. The original medium was replaced by serum free LGM medium without or with M-CSF (100 ng/ml) one hour later. The next day, cell-free supernatants were collected for the SEAP analysis. Cells were stained with Annexin V/propidium iodide (PI). For the inhibition studies, cells were pre-incubated with inhibitor for 30 minutes in X-vivo medium prior to the addition of M-CSF."}