Secretomics screen on LGR4-STF HEK293 cells.

<p>A HEK293 cell line harboring stably integrated LGR4 and the STF reporter construct were incubated with conditioned media of a library of 2859 proteins known or predicted to be secreted. Firefly luciferase values were determined for each sample and normalized to Alamar blue values of the respective samples. Media + PBS and pcDNA DEST40: Negative controls, not expressing any secreted protein; recombinant RSPO1 samples, positive controls using recombinant RSPO1 at concentrations ranging from 0.1−240 ng/ml; RSPO1 pcDNA DEST40, positive control expressing RSPO1 cDNA; Sample: Combined dataset of all secreted (known or predicted) proteins, each protein assessed from conditioned media of duplicate cDNA transfections. RSPO1-4 proteins (black squares) scored highest. Then WNT proteins (red triangles) WNT1, WNT2, WNT3A, WNT7B and WNT8A reproducibly scored above a threshold of 10⁴.⁵ that marks the upper range of the bulk of datapoints, whereas WNT2B, WNT5A, WNT5B, WNT6, WNT8B, WNT9B, WNT10A, WNT10B, WNT11 and WNT16 scored below; WNT3, WNT4, WNT7A and WNT9A centered around the threshold. Each dot, triangle or square represents the luciferase value of a unicate transfected cDNA construct. Shown is an aggregate display of the primary screen and hit confirmation analysis.