Figure 7.

In vivo cleavage of mutant Cas. Rat-1 cells transiently transfected with pFLAG-CMV-5c containing each mutant Cas DNA such as vector only (lane 1), wild-type Cas (wt, lanes 2 and 4), single-mutant Cas (D⁴¹⁶E, lane 5; and D⁷⁴⁸E, lane 6), and double-mutant Cas (D⁴¹⁶E and D⁷⁴⁸E, lanes 3 and 7) were cultured with (lanes 4- 7) or without (lanes 1–3) etoposide for 24 h. The cell lysates were immunoblotted with anti-FLAG M2 mAb (A), and the same membrane was then stripped and reprobed with Cas mAb (B). Note that the overexpresed mutant Cas were resistant to in vivo cleavage. Molecular mass standards are shown by the arrowheads on the left. Molecular masses of the cleavage products are shown by the arrows on the right. Nonspecific binding of antibodies is indicated by closed squares on the right.