|Screen Status:||Primary Repeat|
|Screen Overview:||Genome screen using ectodomain shedding assay|
|Screener:||Marie Kveiborg (Parker Lab).
Extension:3505, Rm 203
|Library:||Full Genome 96|
|Library Description:||270 plates consisting of Kinome, GPCR, druggable and genome libraries|
|Library Format:||267 plate(s) in 96 well format|
|Replicate Info:||2 replicates
|Publications:||The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.. Dombernowsky et al., 2015
|Keywords:||ADAM proteases, HT1080, PKC|
Day 1: Transfection of HT1080 cells stably expressing AP-HB-EGF
HT1080-AP-HB-EGF cells were reverse tranfected with siRNA using INTERFERin transfection reagent in 96-well plates.
Day 3: TPA induction / media change and cell fixation
Plates were washed twice with PBS and treated with 400nM TPA or Serum Free Media (control) for 30mins at 37C.
Media was then removed and cells were fixed in 4% formaldehyde.
Weeks 2 - 6: Immunofluorescent staining of cell surface expression of AP-HBEGF
Plates were stained with:
Acumen Explorer eX3 laser scanning cytometer was used to determine cell number and cell surface expression of AP-HBEGF.