Ripa buffer

293FT cells were transfected with HA Clover KEAP1 WT as described above. After 48 hours, the cells were washed once in ice-cold PBS and collected in ice-cold RIPA buffer (Sigma). The cells were incubated on ice for 30 minutes with intermittent vortexing, and the resultant lysates subjected to centrifugation at 10,000 rpm for 10 minutes at 4 °C. A fraction of the supernatant was reserved for input and prepared for western blotting as described above. The remaining supernatant was exposed to GFP-Trap agarose beads (Proteintech) and rotated end-over-end at 4 °C overnight before washing 5 times with ice-cold RIPA buffer. The beads were then equilibrated in room-temperature binding buffer (25 mM Tris (pH 7.5), 100 mM NaCl, 0.1% NP-40, 1 mM DTT, 5% glycerol). 10 μg of purified His6-KEAP1 was added and the samples were rotated end-over-end at room temperature for 15 minutes. MCB-613 (2, 20, or 200 μM) was added and the samples were rotated for another 15 minutes. Samples were washed 3 times in room-temperature binding buffer, re-suspended in 80 μl of NuPAGE LDS Sample Buffer (4X), boiled at 95 °C for 5 minutes, and analyzed by western blot. Targets were detected using anti-GFP (#2555, CST) and anti-His (#2365, CST) primary antibodies.

Bax, Bcl-2, cleaved caspase-3, cdk6, cytochrome c, cyclin D1, cyclin E1, GAPDH, IκB-α, NF-κB/p65, p21, secondary antibodies, and RIPA buffer were from Proteintech (Wuhan, China). Phospho-IκB-α (Ser 32) primary antibody was purchased from Cell Signaling Technology (Cell Signaling Technology, CST, Beverly, MA, USA). The Annexin-V/PI Kit, mitochondrial membrane potential kit, Hoechst 33258 assay kit, DNA fragmentation kit, and Caspase-3, -8, and -9 kits were purchased from keyGEN BioTECH (Nanjing, China). The nuclear and cytosolic protein extraction kit was purchased from Beyotime (Nanjing, China). The qRT-PCR kit was purchased from Transgene (Shenzhen, China). All other reagents and chemicals were purchased from standard commercial sources.

Spinal cords were homogenized and CD4⁺ T cells were lysed with cold RIPA buffer (Proteintech, IL, USA) supplemented with a protease inhibitor cocktail (Sigma). After centrifugation at 4 °C for 15 min, the supernatants were collected and used for western blot analysis. The following antibodies were used: anti-Bax (1:1,000, Proteintech, IL, USA), anti-MBP (1:1,000, Proteintech), anti-cleaved Caspase 3 (1:1,000, Affinity, OH, USA), anti-cleaved Caspase 9 (1:1,000, Affinity), anti-Bcl-2 (1:1,000, Proteintech), anti-cleaved PARP (1:1,000, Affinity), and anti-β-actin (1:5,000, Bioworld, MN, USA).