Goat anti rabbit secondary antibody sc 2004

About 100 µg of cell lysates were resolved on Bolt™ 4–12% Bis-Tris Plus Gels (NW04120BOX, Thermo-Fisher Scientific). The separated proteins were transferred on to Odyssey^® Nitrocellulose Membranes (LI-COR, Lincoln, NE, USA) using a Mini-PROTEAN^® 3 Electrophoresis System (Bio-Rad, Hercules, CA, USA) filled with 800 mL of transfer buffer and run at 20 V for 10 h at 4 °C using a PowerPac Basic (Bio-Rad). The membranes were blocked with 5% milk in TBST for 1 h. The SRC, EPHB2, and GAPDH bands were probed with specific antibodies and then secondary antibodies. The bands were visualized after being incubated in 5% milk in TBST with 1:2000 goat anti-rabbit secondary antibody (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The membranes were put in between two pieces of plastic wrap and loaded into an Amersham Hypercassette (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Membranes were exposed with HyMembrane CL Autoradiography Film (Denville Scientific, Metuchen, NJ, USA) to obtain images that were subject to scanning and then densitometric quantification for protein expression using ImageJ [85 (link)].

HMECs were seeded at a density of 1,000,000 per well and grown in 6-well plates until they reached full confluence, then cells were incubated with bardoxolone methyl, dimethyl fumarate, and L-sulforaphane for three hours. To measure Nrf2 expression in the nucleus, a Nuclear Extract Kit (Active Motif) was used to isolate the nuclear and cytoplasmic fractions. Nuclear and cytoplasmic extracts (25 or 30 μg protein/sample) were processed as follows: first, the membranes were scanned for total protein content for further band normalization, and subsequently, they were incubated with primary rabbit polyclonal Anti-Nrf2 Antibody (H-300) (sc-13032, Santa Cruz Biotechnology, lot No. GR197455-1) 1 : 1000 overnight and with goat anti-rabbit secondary antibody (sc-2004, Santa Cruz Biotechnology, lot No. 12314) 1 : 2500 for 45 minutes. After chemiluminescent band detection, membranes were washed and incubated with primary Anti-Lamin A/C Monoclonal Antibody (mab636, Thermo Fisher Scientific, lot No. QF215120) 1 : 1000 overnight and with anti-mouse secondary antibody (sc-516102, Santa Cruz Biotechnology) 1 : 5000 for 1 hour. Bands were again detected with the use of chemiluminescence, measured and normalized to total protein content using Image Lab Software (Bio-Rad).