The In-Cell Western™ Assay can be used in a functional siRNA screen measuring the effects of knockdowns in cultured cells.
Hoffmann et al.1 used In-Cell Western screening to assess the effects of knockdowns on mTORC1-dependent phosphorylation of ribosomal protein S6 (rpS6).
Schematic of the workflow for the siRNA screen. (Adapted from Greg Hoffman, Harvard.)
Figure 1. (A) Results of pilot small molecule screen performed with a library of ~2500 known bioactive compounds. Compounds with average Z-score < -2 are considered hits and are shown in red. Known inhibitors of mTORC1 signaling found in the library are shown in green. Star-shaped symbols represent compounds with > 4-fold reduction in cell number. (B) Plate to plate reproducibility is shown for a representative plate from the small molecule library.
1. Hoffmann et al. A functional siRNA screen for novel regulators of mTORC1 signaling. Poster presentation, ASCB Annual Meeting (2008)