Total protein extraction kit

Kidney samples were prepared as descripted in 2.7 for Western blotting. Briefly, total protein was obtained using the Invent Total Protein Extraction Kit (Invent SD001/SN002, Plymouth, USA), and the protein concentration was detected by BCA protein content detection kit (Bioplatform BP104, Nanjing, China). Samples with equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidine difluoride membrane (Millipore, Bedford, MA, USA). After blocking, the membrane was incubated with the primary polyclonal antibody of Zetnf-b (1:1,000), which was prepared as descripted in 2.4 and rabbit polyclonal anti-β-actin (1:1,000; Abcam; UK) at 4°C for 12 h. After washing, the membrane was incubated with goat-anti-mouse (1:8,000; Utibody; Tianjin, China) and Goat Anti-mouse Rabbit (1:8,000; Utibody; Tianjin, China) secondary antibodies for 2 h at 28°C. Detection was performed using an ECL hemiluminescence kit (ThermoFisher, Waltham, MA, USA). After the film was scanned, the gray value of each protein band was measured, and data were presented as a ratio of the value to that for-actin. Zetnf-b expression in the kidney of mock-infected zebrafish was set to 1.0 and marked as 0 h, and the expression of Zetnf-b in infected zebrafish at 2–24 hpi was compared to the expression at 0 h.

Total Protein Extraction kit (Invent Biotechnologies, Plymouth, MN, USA) was used to isolate total protein from tissues or cells. In total, 25 µg of total proteins was suspended in a 5× sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample loading buffer (Beyotime) and boiled at 90°C for 5 min. Next, SDS–PAGE and membrane transfer were sequentially performed prior to antibody incubation for DKC1 (sc-373956, 1:500, Santa Cruz Biotechnology, Shanghai, China), GAPDH (AF5009, 1:5,000; Beyotime), and mouse IgG(H + L) (A0216, 1:1,000; Beyotime). The signals were detected by the ECL-PLUS kit (GE Healthcare, Piscataway, NJ, USA) and MYECL Imager (Thermo Fisher Scientific). Relative DKC1 protein expression was calculated with normalization to GAPDH.

Total protein of testes was extracted with the Total Protein Extraction Kit (Invent) and the protein concentration was examined by BCA method (Thermo). Protein samples were separated by SDS-PAGE and then transferred to PVDF membrane (Millipore). After blocking in 5% milk, the membranes were incubated with indicated primary antibodies at 4 °C overnight. The following antibodies were used: FANCI (Novus, 1:5000), FLAG (Sigma, 1:5000), and α-Tubulin (Proteintech, 1:3000). After being incubated with secondary antibodies, the membranes were detected with an ECL system (Millipore). The ChemiDoc MP System (Bio-Rad) was used to capture images.

Cells were grown to 80% confluency in T-75 flask and then treated with the respective irradiation. Total protein and plasma membrane fraction from cells were isolated using Total Protein Extraction Kit and Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Eden Prairie, MN), respectively. Protein content of the supernatant was determined by the Bradford method with a commercial protein assay reagent (Bio-Rad, Munich, Germany). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with gels containing 10% polyacrylamide. Proteins were transferred to nitro cellulose membranes. Nitro cellulose membrane was incubated for 1h with 5% BSA in Tris-buffered saline (TBS) and probed with the CXCR4 antibody and the anti- Na+-K+ ATPase over night at 4°C in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% BSA. All antibodies were diluted as the recommendation of manufacturer. Membranes were subsequently incubated with secondary antibody conjugated to horseradish peroxidase for 1 hour at room temperature, and immunocomplexes were visualized by enhanced chemiluminescence (Thermo Fisher Scientific). Saos-2 cell lysate (sc-2235) and Jurkat cell lysate (sc-24788), both purchased from Santa Cruz Biotechnology (Heidelberg, Germany), were included in Western blot analysis as negative and positive control for CXCR4 protein expression, respectively.