PMC:3245220 / 43655-44677 JSONTXT

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T20704","span":{"begin":409,"end":413},"obj":"P17252"},{"id":"T20705","span":{"begin":423,"end":426},"obj":"Q04206"},{"id":"T20706","span":{"begin":423,"end":426},"obj":"P21579"},{"id":"T20707","span":{"begin":528,"end":532},"obj":"P17252"},{"id":"T20708","span":{"begin":732,"end":737},"obj":"P09603"},{"id":"T20709","span":{"begin":734,"end":737},"obj":"P04141"},{"id":"T20710","span":{"begin":900,"end":905},"obj":"P09603"},{"id":"T20711","span":{"begin":902,"end":905},"obj":"P04141"},{"id":"T20712","span":{"begin":988,"end":991},"obj":"Q9U6Y4"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"Transient Transfection of RAW 264.7 Cells\nRAW 264.7 cells were seeded in 12-well plates in RPMI medium containing 5% FBS one day prior to transfection, Secreted alkaline phosphatase (SEAP) reporter plasmids pTAL-SEAP or pNF-κB-SEAP were transfected into cells using Qiagene Effectene or Attractene transfection kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. For co-transfection studies, PKCα or NF-κB p65 constructs were mixed at a ratio of 5∶1 with the reporter plasmid. For siRNA transfection, 100 nM of PKCα siRNA or control siRNA were used. Cells were incubated with the transfection reagents for 16–24 hours, washed and starved in RPMI without FBS for 4 hours. Samples were then stimulated with 100 ng/ml M-CSF for 4 hours at which point the medium was collected for the SEAP analysis. For inhibition studies, cells were pre-incubated with inhibitors for 30 minutes before M-CSF treatment. On average, we realized 30–40% transfection efficiency as confirmed by GFP expression in Raw 264.7 cells."}

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