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Additional counter screening for autofluorescence identified another false-positive (pyrvinium pamoate).
CCY generated the LNCaP-NKX3.1-YFP cell line used for counter screening and assisted with advice.
After hit validation and counter screening one hit compound, VLX40, was selected for mechanistic investigation and further preclinical evaluation.
Counter screening was performed against three bacterial-type chitinases: A. fumigatus chitinase B1 [ AfChiB], acidic mammalian chitinase [AMCase] and H. sapiens chitinase 1/chitotriosidase [ HsCHT]).
She developed the screening assay, performed all screening and counter screening, carried out the bulk of the validation assays and drafted the manuscript.
Further prioritization of the remaining 15 SMIPs was achieved by counter screening against another unstable prostate tumour suppressor (NKX3.1) [ 41, 42].
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In confirmatory screen AID1891 the protocol states: Counter screen for luciferase inhibitors of Dense Granule Secretion.
However, antibodies derived from in vitro selection or maturation lack an in vivo-like control system and rely on the counter screen to ensure specificity.
Following confirmation, counter screens, and dose response analysis, we prioritized more than 100 compounds for further in vitro and in vivo analysis.
In the counter screen, parental MCF-10A cells cultured on polyhema plates were transfected with siRNA duplexes.
In brief, hybridomas were generated using standard techniques, tested for specificity to myostatin and counter screened for growth and differentiation factor-11, transforming growth factor-β1, activin A and BMP-5 binding.
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