To detect TYLCCNV CP in whiteflies, samples of 200 whiteflies each were prepared using radioimmunoprecipitation assay buffer with proteinase inhibitors. Protein samples were separated using 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto PVDF membrane. The membrane was blocked with 5% milk in TBS (10 mM Tris HCl, 150 mM sodium chloride, pH 7.5), and then incubated with appropriate primary antibodies. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, blots were visualized with the ECL Plus Detection System (Bio-Rad). Antibodies to TYLCCNV CP (IC4) were kindly provided by Professor Jianxiang Wu, the Institute of Biotechnology, Zhejiang University. Commercial antibodies to ACTIN (beta-actin) were purchased from EARTHOX life sciences.
To detect the expression of HES1 in S2 cells, cells were washed twice using PBS and then re-suspended with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, pH 7.8). After centrifugation, the supernatant was boiled in PAGE buffer for 5 min. The presence of target proteins was verified using western blotting with an anti-His monoclonal antibodies (Abcam).
To detect the expression of HES1 in S2 cells, cells were washed twice using PBS and then re-suspended with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, pH 7.8). After centrifugation, the supernatant was boiled in PAGE buffer for 5 min. The presence of target proteins was verified using western blotting with an anti-His monoclonal antibodies (Abcam).