Coculture with BMSCs does not protect against INK128-induced MM-cell cytotoxicity. (A) Detection of Erk, 4EBP1, and Akt activation with the nanofluidic proteomic immunoassay in response to coculture with BMSCs, IL-6, or IGF1 in MM1S and OPM2 cells. Peaks on the traces that represent phosphorylated isoforms are indicated with black arrows. All measurements were performed in triplicate. (B) MM1S and OPM2 cells were cultured with rapamycin (10-100nM) or INK128 (10-100nM) for 48 hours in the presence or absence of BMSCs. Cell proliferation was assessed using the BrdU uptake assay. (C) MM1S and OPM2 cells were cultured with rapamycin (25-100nM) or INK128 (25-100nM) for 5 hours in the presence or absence of BMSCs. Whole-cell lysates were subjected to Western blotting using anti–p-rS6, anti–p-4EBP1, anti–p-Akt, and anti–α-tubulin Abs. (D) MM1S and OPM2 cells were cultured with rapamycin (25-100nM) or INK128 (25-100nM) for 5 hours in the presence or absence of IL-6. Whole-cell lysates were subjected to Western blotting using anti–p-rS6, anti–p-4EBP1, anti–p-Akt, and anti–α-tubulin Abs.