Analysis of other factors involved in sensitivity to TRAIL: PKC, AKT, ERK, and NFκB. KOB cells (5 × 10⁵/mL) were incubated with 0.5 μg/mL TRAIL [T], 12.5 μg/mL BB-1 [B], BB-1+TRAIL [B+T], or without reagents [C]. (A) PKC activity. Cells were incubated for 24 hours, and PKC activity was evaluated. ▪, results relative (%) to the value for untreated cells; □, cells treated without ATP. The assay was performed in triplicate, and data are the mean ± SD. (B) Western blot analysis of AKT and ERK 1/2. Cells were harvested at the indicated time points, and Western blotting was performed using 30 μg cell extract. (C) Activities of NFκB transcription factors. Cells were incubated for 24 hours, nuclear extracts were prepared, and activities of p50, p65, c-Rel, and p52 were evaluated. Raji cells were used as a positive control (PC) (□). The fold activation (▪) was obtained by setting the value for the positive control cells as 1.0. The assay was performed in triplicate, and data are the mean ± SD.