Author: Michael T. Parker; Smita Gopinath; Corey E. Perez; Melissa M. Linehan; Jason M. Crawford; Akiko Iwasaki; Brett D. Lindenbach
Title: Innate Immune Priming by cGAS as a Preparatory Countermeasure Against RNA Virus Infection Document date: 2018_10_3
ID: j1gkl43g_4
Snippet: were used in these experiments because this cell line lacks endogenous cGAS 174 expression and could be reconstituted with WT or mutant hcGAS-HA3x; however, 175 unlike MEFs and THP-1 KO cells, HEK 293E cells could be efficiently transfected 176 with DNA and readily scaled up for isolation of cGAMP from cytosolic extracts. As 177 shown in Figure 4A , a unique UHPLC peak (~5 minutes elution) was observed after 178 transfecting WT hcGAS-HA3x-express.....
Document: were used in these experiments because this cell line lacks endogenous cGAS 174 expression and could be reconstituted with WT or mutant hcGAS-HA3x; however, 175 unlike MEFs and THP-1 KO cells, HEK 293E cells could be efficiently transfected 176 with DNA and readily scaled up for isolation of cGAMP from cytosolic extracts. As 177 shown in Figure 4A , a unique UHPLC peak (~5 minutes elution) was observed after 178 transfecting WT hcGAS-HA3x-expressing HEK 293E cells with salmon sperm DNA; 179 MS analysis confirmed that this peak corresponded to cGAMP (Figures 4B and 4C) . published cGAMP bioassays (21). Again, we were unable to detect cGAMP in 194 lysates from VSV-infected or SINV-infected THP-1 cells expressing WT hcGAS, 195 while a synthetic cGAMP control led to robust phosphorylation of IRF3 ( Figure 4I ). 196
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