Horseradish peroxidase linked secondary antibody

Brown planthopper was treated with schaftoside by feeding the nymphs with artificial diet as described above. Western blot was performed according to the method modified from Hao et al. (2015) (link). At day 10, about 25 nymphs of each biological replicate were homogenized in 1 × PBS, and then added 2 × SDS sample buffer. The lysate protein were boiled for 5 min, and centrifuged at 10,000 × g for 5 min. Protein samples of 10 μl were loaded onto the SDS gels, electrophoresis for 90 min, and transferred to polyvinylidene difluoride membranes. TBST (0.1% Tween 20 in TBS) and 5% non-fat powdered dry milk (w/v) were used to block the blots for 2 h at room temperature. Primary antibody anti-phospho-CDK1 (pThr14, Sigma-Aldrich, MO, United States) at a dilution of 1:500 was then incubated with the blot for 12 h at 4°C in a TBST solution to probe pThr14 of NlCDK1. The membrane was washed with TBST for 10 min 3 times, and then incubated in the horseradish peroxidase-linked secondary antibody (Solarbio, Beijing, China) at a dilution of 1:1000. The membrane was washed 3 times, detected using the DAB Horseradish Peroxidase Color Development Kit (Sangon Biotech, Shanghai, China), and imaged with a camera equipped in a mobile phone.