Sc 2055

HRP goat anti-mouse IgG (RRID: AB_631736; 1:8000; sc-2055; Santa Cruz, Heidelberg, Germany).
HRP goat anti-rabbit IgG (RRID: AB_631748; 1:8000; sc-2054; Santa Cruz, Heidelberg, Germany).

Cells were lysed and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked and incubated with primary antibodies (Table S1) at 4 °C overnight, followed by incubation with a secondary antibody (1:2000, Cat.#sc-2055, Cat.#sc-2004, Santa Cruz, CA, USA). The membranes were developed using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Equal sample loading was confirmed by probing the membranes with GAPDH. The intensity of the protein bands was determined via densitometry using ImageJ system.

The whole cell lysates were prepared by using RIPA Lysis and Extraction Buffer (Thermo Scientific, IL, USA) with Pierce Protease and Phosphatase Inhibitor Mini (Thermo Scientific, IL, USA). The fractionated lysates were prepared by using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific, IL, USA). Protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad; CA, USA), and the concentration in individual samples was equalized before adding 4x Laemmli buffer to a final concentration of 1x. Equal amounts of protein (50 μg whole cell lysates, 45 μg fractionated lysates) were run on 10% SDS-PAGE gels and then transferred with iBlot 2 Transfer Stacks and iBlot 2 Dry Blotting System onto PVDF membranes (Invitrogen, Thermo Scientific, IL, USA). After one hour 5% milk block, membranes were probed with the antibodies diluted with 5% BSA (NRF3) or 5% milk (β-actin and PCNA and secondary antibodies) in Tris-buffered saline with 0.1% Tween 20. Primary antibodies anti-NRF3 (HPA055889, Sigma-Aldrich), anti-β-actin (NB600-501, Novus Biologicals, UK), and anti-PCNA (NB600-501SS, Novus Biologicals, UK) were incubated overnight, and appropriate HRP-conjugated secondary antibodies (sc-2054 and sc-2055, Santa Cruz, CA, USA) were incubated at RT for one hour. Blots were detected with the Western blot imaging system Azure 600 (Azure Biosystems, CA, USA).