Fig. S6.

Analysis of the in vivo stability of different flavoproteins by cycloheximide chase. HEK293T cells were electroporated with wild-type NQO1, the P187S mutant (MUT), Δ50-NQO1 (WTΔ50), or the Δ50-NQO1-P187S mutant (MUTΔ50), and NQO2 expression plasmids and were incubated in normal (A) or riboflavin-deficient (B) medium. Four hours later, half of the cells were collected and lysed; 1 mM cycloheximide (CHX) was added to the other half to stop protein synthesis for an additional 4 h. A portion of the wild-type NQO1-transfected cells was kept without cycloheximide to document the ongoing translation (first lanes in A and B). (Upper) Amounts of flavoproteins were determined using Western blotting. (Lower) Lysate loading was controlled by Ponceau staining. One representative of two experiments is shown.