Quantitative RT-PCR and Immunoblot Analysis of NRF2 and FN3K Pathways

Quantitative RT-PCR (qPCR) and immunoblots were performed as before [14 (link)]. We used the following taqman probes for qPCR analysis: HUMAN—NRF2 (Hs00975961_g1, Life Technologies, Carlsbad, CA, USA), FN3K (Life Technologies, Hs_00223368_m1), GUSB (Life Technologies, 4333767 F), and ACTB (Life Technologies, 4332645); MOUSE—Nqo1 (Life Technologies, Mm01253561_m1), Nrf2 (Life Technologies, Mm00477784_m1), Fn3k (Life Technologies, Mm00445584_m1), cMyc (Life Technologies, Mm00487804_m1) and Actb (Life Technologies, Mm00607939_s1). For immunoblotting, we used the following antibodies from Cell Signaling (Danvers, MA, USA): NQO1 (CST, 62262), MYC (CST, 5605), BCL2 (CST, 3498), KEAP1, (CST, 8047), CUL3 (CST, 2759), GAPDH (CST, 5174), and β-actin (Sigma-Aldrich A5441 St. Louis, MO, USA). Proteins were visualized using LI-COR Lincoln, NE, USA detection system after incubation with following secondary antibodies: Goat anti-rabbit-IR800 (LI-COR, 926–32211) and Goat anti-mouse-IR680. We used the following reporter assays as per manufacturer’s instructions: Luciferase-Glo (Promega) and Beta-glo (Promega). T7 endonuclease assays were conducted to confirm gene editing (NEB, Ipswich, MA, USA).