Figure 5.

NEDD4L accelerated the ferroptosis of GCs by promoting the ubiquitin-mediated proteasome degradation of GPX4. (A) NEDD4L interacts with GPX4 directly. The protein lysis from GCs was immunoprecipitated with IgG or antibodies against NEDD4L or GXP4 and then IB with NEDD4L and GXP4 (n = 3). (B and C) GPX4 protein stability is regulated by NEDD4L. GCs with or without NEDD4L knockdown were treated with 15 µg/mL cycloheximide (CHX) for various times, as indicated by the GPX4 protein stability assessed by western blot (n = 3). **P < 0.01. (D) NEDD4L regulates GPX4 stability through proteasome-mediated ubiquitination and degradation pathway. GC cells with or without NEDD4L-OE were treated with MG132 (20 μM) and then analyzed by western blotting. (E) Cell lysates were extracted from scramble and si-NEDD4L-infected GCs and immunoprecipitated with anti-GPX4 antibody and then analyzed by western blot using anti-ubiquitin antibody (n = 3).