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 Supplier: Dharmacon
Library Format: 267 plate(s) in 96 well format
Replicate Info: 2 replicates
Technical 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
Cell Line: HT-1080

Screen Method

Untreated

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:

Readout

Acumen Explorer eX3 laser scanning cytometer was used to determine cell number and cell surface expression of AP-HBEGF.