Ecl plus detection system

At the end of indicated treatment, preparation of total cellular proteins. The Western blot analyses of these samples were performed as previously [34 (link)]. After blocking with 5% BSA in fresh TBS buffer containing 0.05% Tween-20 at 37 °C for 2 h, the membranes were probed with the first antibody including rabbit anti-Nrf2, anti-NQO1, anti-β-actin antibody, at 4 °C overnight following 1 h incubation in corresponding secondary antibodies (dilution, 1: 5000) at room temperature. The blot in membranes was visualized by an enhanced chemiluminescence (ECL) Plus detection system (P1010, Applygen, Beijing, China). The blots were quantified by Quantity One software (Bio-Rad, Hercules, CA, USA). The concentration value of each cellular protein expression was expressed as each normalized data relative to control.

For the immunoblot analyses, the total protein was extracted from the cytoplasm of the IPEC J2 cells, and the samples were denatured in 4×SDS-PAGE loading buffer (40 mM Tris-HCl, PH 8.0, 200 mM DTT, 4% (v/v) SDS, 40% (v/v) Glycerol and 0.032% (v/v) Bromophenol Blue) (No. 7173 Takara, Ostu, Japan) and boiled for 10 min. The denatured proteins were separated using 12% SDS-PAGE and were transferred onto a PVDF membrane (0.45 μM) (Millipore, Boston, Massachusetts, USA). The membrane was blocked in Tris Buffered Saline with Tween (TBST) (10 mM Tris, 100 mM NaCl, 0.1% Tween 20) with 5% (w/v) nonfat milk powder for 1.5 h. After washing with TBST for three times, the blocked membranes were incubated with the primary antibodies overnight at 4°C in TBST and were then washed three times followed by incubation with the corresponding HRP-linked secondary antibodies (1:2500) for 1 h at room temperature. The membranes were washed three times, and bound antibodies were detected using an ECL plus detection system (P1010, Applygen, Beijing, China). The expression of each protein was normalized to that of β-actin.

Cells were harvested with RIPA buffer with protease inhibitor and phosphatase inhibitor. Protein concentration was determined by BCA assay (Bio–Rad Laboratories, Hercules, CA). Twenty micrograms of protein was resolved by SDS/PAGE, and then transferred on to PVDF membrane. Then the membrane was blocked with 5% BSA for 1 h at room temperature, and hybridized with antibodies of FRα (1:1000), E-cadherin (1:500), TSLC1 (1:1000), β-actin (1:1000), ERK1/2 (1:1000), MEK (1:500), p-MEK (1:500), or p-ERK1/2 (1:1000) at 4°C overnight. The membrane was incubated with horseradish peroxidase conjugated secondary antibody (Zhong Shan Biotech Co. Ltd, Beijing, China) for 1 h at room temperature. The blots were developed by ECL Plus detection system (Applygen Technologies Inc, Beijing, China), and protein bands were analyzed with the software Quantity One (Bio–Rad, Hercules, CA, U.S.A.).