β actin af5003

For each experiment, 50–100 k events from phagocytic hemocytes (R1) and control hemocytes (R2) were collected. Total RNA from the collected samples was purified using the RNAprep Pure Micro Kit (TIANGEN, Beijing, China) and reverse-transcribed into cDNA using a First Strand cDNA Synthesis Kit (Beyotime, Shanghai, China). qPCR was performed as previously described (Luo et al., 2022 (link); Supplementary file 10), and the gene expression level was recorded as relative expression to EF-1α. This experiment was repeated five times. Total proteins from sorted hemocytes were precipitated by adding 1/100 volume of 2% sodium deoxycholate (Macklin, Shanghai, China) and 1/10 volume of 100% trichloroacetic acid (Macklin), followed by vortexing and centrifugation at 15,000×g for 15 min at 4 °C. The pellet was collected for performing SDS-PAGE and immunoblotting, as described before (Luo et al., 2022 (link)). This experiment was repeated thrice. The following antibodies were used: β-actin (AF5003; Beyotime, Shanghai, China), anti-NAGA (13686-T24; SinoBiological, Beijing, China), and anti-LYZ1 (bs-0816R; Bioss Antibodies, MA, USA). The polypeptide antibody against shrimp NLRP3 (aa29-42) was prepared by GenScript (Nanjing, China).

For the apoptosis analysis of the cells after treatments, the primary antibodies used were as follows: Cleaved caspase-3 (Asp175, #9661), γH2A.X (Phospho-Histone H2A.X, Ser139, #9718) and p53 (#2524) antibodies were purchased from Cell Signaling Technology (CST, United States); caspase-3 (AF1213), PARP (AF1657), cleaved PARP (AF1567), Ki67 (AF1738), and β actin (AF5003) antibodies were obtained from Beyotime.
To explore the activation of the NER signal pathway, the primary antibodies used were as follows: ERCC1 (#12345), ERCC4/XPF (#13465), and XPC (#12701) antibodies were obtained from CST; ERCC3/XPB (sc-271500) antibody was purchased from Santa Cruz Biotechnology (United States); ERCC2/XPD (10818-1-AP), ERCC5/XPG (67055-1-Ig), and XPA (16462-1-AP) antibodies were purchased from Proteintech (WUHAN SANYING, China). Protein expression levels were expressed relative to β actin by calculating band intensity values. Anti-mouse IgG and anti-rabbit IgG were used as HRP-conjugated secondary antibodies.

Radioimmunoprecipitation assay (RIPA, R0010) with 1% phenylmethanesulfonyl fluoride (PMSF) buffer was purchased from Solarbio Life Science (Beijing, China). The following primary antibodies were used for the Western blotting of colon tissue homogenate: Claudin-1(A11530) antibody (abclonal, Wuhan, Hubei, China), phosphorylated p38 (p-p38, 4511) and phosphorylated NF-κB (p-p65, 3033) antibodies (Cell Signaling, Danvers, MA, USA), Bax (AF0057), Inhibitor kappa B-α (IκB-α, AF1282), NF-κB (p65, AF0246), PCNA (AF1363), and β-actin (AF5003) antibodies (Beyotime Institute of Biotechnology, Shanghai, China).