Ab205718

Briefly, NPC cells were collected and then lysed using lysis buffer for extraction of total proteins. Next, the proteins were separated on dodecyl sulfate, sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE, 10%), transferred to PVDF members (Millipore, IPVH00010), and incubated using primary antibodies (1:1000; Abcam, Cambridge, MA, USA) against ELF2 (ab225958) and β-actin (ab8227). Finally, the proteins were incubated with the second antibodies (Abcam, ab205718) and analyzed by the ECL plus Kit (Beyotime, Haimen, China, P0018S).

Total proteins were extracted using RIPA reagent (Beyotime), and their concentrations were determined using BCA kit (Beyotime), proteins were loaded to 10% SDS-PAGE for band separation. Separated bands were transferred to PVDF membranes and then blocked with 5% skim milk at 37 °C for 1 h. After that, these membranes containing protein bands were exposed to the primary antibodies overnight at 4 °C environment, such as anti-GPX4 (ab262509, 1:1000; Abcam, USA), anti-SLC7A11 (ab275411, 1:1000), anti-NLRP3 (ab263899, 1:1000), anti-cleaved caspase-1 (ab25901, 1:1000), anti-IL-1β (ab254360, 1:1000), anti-GSDMD-N (ab215203, 1:1000) and anti-GAPDH (ab8245, 1:1000). Next, these membranes were exposed to the corresponding secondary antibody conjugated with horseradich peroxidase (ab205718, 1:2000) at 37 °C for 2 h. Finally, the protein signaling was checked using the enhanced chemiluminescence (ECL) reagent (Beyotime, China), and the intensity of the protein bands was quantified using Image J software 10.0 (Bethesda, USA).