Mouse anti cytochrome c

Cells were lysed in Laemmli sample buffer and run in 4–20% Mini-PROTEAN TGX precast protein gels (Bio-Rad Laboratories). The primary antibodies used were rabbit anti-Egr1 (no. 4154; Cell Signaling), rabbit anti–c-Fos (no. 2250; Cell Signaling), rabbit anti–c-Jun (no. 9165; Cell Signaling), mouse anti-Snail (no. 3895; Cell Signaling), rabbit anti-PARP1 (no. 9532; Cell Signaling), rabbit anti-Smad2/3 (no. 8685; Cell Signaling), rabbit anti-pSmad2/3 (no. 8828; Cell Signaling), mouse anti–cytochrome c (no. sc-13560; Santa Cruz Biotechnology), mouse anti-Cox4 (no. 11967; Cell Signaling), rabbit anti–caspase 9 (no. 9502; Cell Signaling), mouse anti–caspase 8 (no. 9746; Cell Signaling), rabbit anti-Tubulin (no. 2128; Cell Signaling), and mouse anti–α-Tubulin (T6199; Sigma). The secondary antibodies used were IRDye 800CW donkey anti–rabbit IgG (H+L), IRDye 680LT donkey anti–mouse IgG (H+L), and IRDye 800CW donkey anti–mouse IgG (H+L; LI-COR Biosciences). The blots were scanned on an Odyssey imaging system (LI-COR Biosciences). Cell treatment, sample collection, and Western blotting were repeated at least three times, and the representative blots are shown in the figures.

Cells were fixed with 4% formaldehyde, permeabilized with phosphate-buffered saline (PBS)-0.01% NP40, and incubated for 1 h at 37 °C with rabbit anti-phosphoS40-NRF2 antibody (Abcam, 1:100), mouse anti-cytochrome c (Santa Cruz Biotechnology, 1:300) or anti-Hsp60 Alexa Fluor 647 (BD Biosciences, 1:100) followed by Alexa Fluor 488-conjugated chicken anti-rabbit or goat anti-mouse antibody (Thermo Fisher Scientific, 1:1000) for 45 min at 37 °C. Samples were mounted with an anti-fade reagent (Prolong Gold, Molecular Probes) after a 10-min incubation with Vybrant DyeCycle Ruby Stain (Thermo Fisher Scientific, 1:1000 in PBS) to visualize the nucleus. Images were obtained with a Zeiss LSM510 or Zeiss Airyscan LSM900 confocal microscope. Data were analyzed with ZenBlue software.