Ku86 truncation is not the result of in vitro generated proteolysis in human MM cell lines. Immediate whole cell lysis (5.0 × 106 cells/sample) was performed on freshly-obtained human PB T cells (sample from patient 1), CESS, K562, HL60, and RPMI 8226 and SGH-MM5 MM cell lines using a denaturing and reducing gel-loading buffer (95°C for 10 mins) (A). Cell extracts were also prepared using conventional methods from previously frozen and stored human PB T cells (samples from patients 2 and 3), K562 CML, and HL60, and RPMI 8226 and SGH-MM5 MM cell lines (B). Cell lysates (20.0 μg/sample) were resolved on a 12.5% SDS-PAGE gel, transferred onto PVDF membranes, and probed with S10B1 anti-Ku86 mAb, which recognizes the N-terminus of Ku86. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Experiments were performed in triplicate.