Figure 5
From: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
Trypsin digestion generates a 69-kDa Ku86 protein from a full-length cloned human MM Ku86 protein (rhKu86) in vitro. Full-length rhKu86 was first expressed and purified from COS cells. Serial digestion (A) of rhKu86 (6.5 μg/sample) by trypsin (0.065 μg of Trypsin Gold/reaction up to 20 mins) was then performed using a limited proteolysis protocol from the manufacturer. The reactions were stopped by rapid cooling on ice; and the products of trypsin digestion were resolved by SDS PAGE and immunoblotted S10B1 anti-Ku86 mAb. The effect of protease inhibitors aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) on trypsin digestion was also determined on a larger sample of rhKu86 (13.0 μg/sample). Trypsin digestion (0.065 μg of Trypsin Gold/reaction for 1 min) of rhKu86 was performed as above either without (lane 1) or with (lane 2) protease inhibitors (B).