Effects of drug wash-out on protein phosphorylation. (a). MM1s cells were treated with DMSO or GDC-0941 at indicated doses for 2 hours. Cell pellets were formalin fixed and paraffin embedded, and sectioned for pS6 staining (top). The level of pS6 was quantified by IHC and MSD assays (bottom). (b). Two MM cell lines (MM1s and RPMI8226) were treated with DMSO or GDC-0941 (200nM) overnight, washed three times and cultured in regular culture medium in the absence of GDC-0941 or DMSO. Cells were harvested at indicated times for MSD biochemical assay. The ratio of the normalized pS6/tS6 between GDC-0941 and DMSO treatments was used to calculate the percentage of pathway modulation. Data shown (a-b) are representative of at least three independent studies.