Nrf2 polyclonal antibody

H1975 cells were seeded on 12-well plates containing 12-mm coverslips for 24 h before drug treatment. After 30 min or 24 h of drug treatment, the cells were washed twice with PBS Ca²⁺ Mg²⁺ and fixed with paraformaldehyde 4%, followed by PLA. The protocol of PLA was according to Chen and Huang [57 (link)]. Primary antibodies KEAP1 monoclonal antibody (1F10B6, Invitrogen) and NRF2 polyclonal antibody (ab31163, Abcam) were used at a dilution of 1:800. The secondary antibodies attaching probes were provided from the kit Duolink In Situ PLA Probe Anti-Rabbit MINUS (DUO92006, Sigma-Aldrich) and Probe Anti-Mouse PLUS (DUO92001, Sigma-Aldrich). The protein-protein interactions (PPIs) were detected with detection reagent FarRed (DUO92013, Sigma-Aldrich). Phalloidin-Alexa 488 nm and DAPI were used to visualize the cytoplasm and nucleus, respectively. Images were taken by using a spinning disk confocal microscopic system with a magnification of 60× objective. Total number of blobs was calculated as Z-projection via Metamorph software. A student’s t-test was applied to compare the difference between the control and each treatment group.

Snap-frozen lung tissues were resuspended in protein lysis buffer and sonicated. Homogenates were vortexed briefly, incubated on ice for 10 min, for a total of 3 times. Samples were centrifuged at 13,500 RPM at 4 °C and supernatants were collected to determine protein concentration via Bradford assay. Proteins were separated on a 10% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on to a nitrocellulose membrane. After blocking with 5% skim milk, membranes were probed using catalase (CST 14097), p65 NF-κB (CST 6956), phospho-p65 NF-κB (CST 3033), NQO1 (CST 3187) monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA), Nrf2 polyclonal antibody (ab31163; Abcam, Cambridge, UK), superoxide dismutase 1 (SC-101523), and beta-actin (SC-47778) monoclonal antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and incubated overnight at 4 °C. HRP-conjugated anti-mouse IgG and anti-rabbit IgG were used as secondary antibodies, then expressions were detected on an x-ray film in the darkroom following enhanced chemiluminescence (ECL) exposure. Densitometry analyses of Western blot images were performed using the ImageJ software.