Pvdf membranes

Thirty μg of cell lysates were separated by 10% SDS-PAGE under reducing conditions and transferred to PVDF membranes (Cwbiotech, China). Primary mouse anti-GFP antibody (Cell Signaling Technology, Danvers, MA, United States) at a dilution of 1:1000 was used to probe proteins. Membranes were then incubated with a fluorescent secondary antibody. The Odyssey^® LI-COR Imaging System (LI-COR Biotechnology, Lincoln, NE, United States) was used to visualize proteins.

The A549 cells were collected 60 h post of infection. The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitors (Beyotime, Shanghai, China) added to collect total proteins. The proteins were loaded onto SDS-PAGE gels (10–15%), electrophoresed, and then transferred to polyvinylidene difluoride (PVDF) membranes (CWBiotech, Beijing, China) through an electrophoretic transfer chamber (Millipore, Temecula, CA, USA). The membranes were washed with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) three times and blocked with 5% nonfat milk in TBST at 37 °C for 2 h. Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature (Goat anti-Rabbit IgG: Abcam, ab205718, 1:5000 dilution). The primary antibodies used were: GAPDH: CST, 5174, 1:1000 dilution; IRF1: CST, 8478, 1:1000 dilution. Finally, the immunoblot bands were visualized with an enhanced chemiluminescence (ECL) kit (Beyotime, China) and read using a chemiluminescence system (Thermo Scientific, Waltham, MA, USA).

The antibody of BtFAD2‐9 protein used for Western blots was generated from synthetic peptides (Pujian Biotech) derived from respective specific amino acid sequences ³¹⁶HHLFPTMPHYHAVEAC³³⁰, and other specific antibodies targeting β‐actin and β‐tubulin were commercially purchased (Abcam, ab115777 and ab18207). The protein level of target proteins was determined with Western blots using β‐actin or β‐tubulin as internal controls. The protein samples (ca 30 µg protein extracted from whitefly and Drosophila mixed adult simple, respectively) were isolated using 10% SDS‐PAGE and transferred onto PVDF membranes (Merck Millipore). The PVDF membranes were then blocked with blocking buffer containing BSA (CWBIO) at 25 °C for 1 h and incubated with the appropriate primary antibody (1:5000) at 4 °C overnight, followed by incubation with goat anti‐rabbit horseradish peroxidase‐conjugated secondary antibody (1:5000, CWBIO). The protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific), and the images were captured by the Tanon‐5200 Chemiluminescent Imaging System (Tanon). Densitometric analysis of the protein bands was performed using ImageJ v.1.51 software (https://www.rsbweb.nih.gov/ij/), and the relative band intensities were calculated based on densitometric ratios between target proteins and internal controls.