Western Blot Immunodetection Protocol

Western blot was performed as previously reported^{44 (link)}. Briefly, the membranes were incubated overnight at 4 °C with the following corresponding primary antibodies: anti-HECTD1 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HECTD2, anti-HECTD3 (1:800, BIOS, Beijing, China), anti-TRAF3 (1:200, Affinity Biosciences, Shanghai, China), anti-phospho-IKKβ (Tyr199), anti-phospho-IκBα (Ser32/Ser36), anti-phospho-p65 (Ser536) (1:500, Affinity Biosciences, Shanghai, China), anti-IKKβ, anti-IκBα, anti-p65 (1:1000, Proteintech, Wuhan, China), anti-GST tag, anti-His tag, anti-FLAG tag, anti-HA tag (1:5000, Proteintech, Wuhan, China), and anti-β-actin (1:3000, Proteintech, Wuhan, China) antibodies.