Ubiquitin

To generate the GST‐tag fusion protein of ZmKW1, the full‐length CDS of ZmKW1 was amplified and cloned into a PGEX‐4 T‐1 vector using EcoRI and SalI enzyme sites (TransGen Biotech, Beijing, China). The constructed vector was then transformed into a Transetta (DE3) Chemically Competent Cell (TransGen Biotech) for protein expression. Following induction with 0.5 mM IPTG, protein purification was performed with the GST 4FF Sefinose Resin Kit (Sangon Biotech, Shanghai, China). The purified protein was concentrated via ultrafiltration and quantified using BSA as a standard. To verify the ubiquitase activity of ZmKW1, a system was set up consisting of 500 ng of purified GST‐ZmKW1, 10 μg of recombinant Ub (Sigma, Darmstadt, Germany), 0.1 μg of rabbit E1 (Boston Biochemicals, Boston, MA), 0.2 μg of E2 UbcH5b (Boston Biochemicals), 2 mm ATP, 50 mm Tris–HCl (pH 7.4), 5 mm MgCl₂, and 2 mM DTT. After incubation at 30 °C for 2 h, the reaction was terminated by adding 2× SDS‐PAGE loading buffer at 95 °C for 5 min. Ubiquitinated proteins were detected by Western blotting using anti‐GST (Abclonal, Wuhan, China) and anti‐Ubiquitin (Abclonal) antibodies. The protein was detected with GST (1:5000) and Ubiquitin (1:2000) at 4 °C overnight, followed by secondary anti‐mouse‐HRP at a concentration of 1:5000 (Abclonal). Imaging was done with a Tanon‐5200 system (Tanon, Hangzhou, China).

Lipopolysaccharide (LPS) (Sigma, St. Louis, MO, USA), peptidoglycan (PGN) (Invivogen, San Diego, CA, USA), Poly(I:C) (Invitrogen, Carlsbad, CA, USA), R837 (Invivogen), CpG (Invivogen), murine IL-1β (PeproTech, Rocky Hill, NJ, USA), SYBR (Bio-Rad, Hercules, CA, USA), TRIzol reagent (TaKaRa, Dalian, China); mouse monoclonal antibodies against Flag (Sigma), HA (Covance, Princeton, NJ, United States), Myc (Proteintech, Rosemont, IL, USA), β-actin (Proteintech), IκBα (CST, Danfoss, MA, USA) and phospho-IκB (Ser32/36) (CST); rabbit monoclonal antibody against MyD88 (CST), phospho-IRAK4 (Ser345/346) (CST) and phospho-IRF3 (Ser396) (CST); rabbit polyclonal antibody against IRF3 (Proteintech), IRAK1 (CST), IRAK2 (CST), IRAK4 (CST), ubiquitin (CST), K48-polyubiquitin (CST), H3 (ABclonal, Wuhan, China), and SPOP (Proteintech) were purchased from the indicated manufactures.