Figure 5.

NQO1 increases c-FOS stability. (A-B) RKO/pshCont and RKO/pshNQO1 cells (A) and MDA-MB-231/pNQO1 and MDA-MB-231/pCont cells (B) were incubated with or without EPX for 1 h in the presence or absence of CHX. Whole cell lysates were immunoblotted for c-Fos, NQO1, and β-actin. (C) MDA-MB-231 cells were transfected with pNQO1-myc-His₆ and pEGFP-c-Fos. Whole-cell extracts were immunoprecipitated with anti-His₆, anti-GFP and anti-IgG (negative control) and analyzed by immunoblotting with anti-NQO1 anti-c-Fos antibodies. (D) Whole-cell extracts of RKO/pshCont cells were immunoprecipitated with anti-NQO1, anti-c-Fos and anti-IgG (negative control) and analyzed by immunoblotting with anti-NQO1 anti-c-Fos antibodies. (E) (Upper panel) Illustration of domains of c-Fos. DBD, LZ, and TAD represent DNA-binding domain, leucine zipper domain and transcription activation domain, respectively. (Lower panel) MDA-MB-231 cells were transfected with pNQO1-myc-His₆ and pEGFP-c-Fos expressing deletion constructs, subjected to Ni-NTA bead-based pulldown assays and analyzed by immunoblotting with anti-NQO1 and anti-GFP antibodies. (F) MDA-MB-231 cells were transfected with pNQO1-myc-His₆ and pEGFP-c-Fos expressing deletion constructs, subjected to Ni-NTA bead-based pulldown assays and analyzed by immunoblotting with anti-NQO1 and anti-c-GFP antibodies. (G) Binding assay of NQO1 and DBD of c-Fos. Samples were reacted and subjected to electrophoresis using reducing or non-reducing SDS-PAGE and silver staining. (H) RKO/pshCont cells were treated with DBD peptide. After 48 h of incubation, whole cell lysates were immunoblotted for c-Fos, and β-actin.