10 kda molecular weight cutoff spin filter

The whole body H₂O₂ concentration in pea aphid was determined as described previously [19 (link) and 67 (link)]. The pea aphids were collected in 50 mM sodium phosphate buffer (pH 7.4) containing 2 mg/mL catalase inhibitor 3-amino-1, 2, 4-trizole (Sigma, St. Louis, MO, USA). After homogenization, the samples were filtered through a 10-kDa molecular weight cutoff spin filter (Millipore, Billeeica, MA, USA). The eluent was collected and the H₂O₂ concentration was measured with a Hydrogen Peroxide Assay Kit (Invitrogen, Carlsbad, CA, USA) on a fluorescence microplate reader (Tecan, Männedorf, Switzerland) according to the manufacturer’s protocol. The values were normalized to the total amount of protein in the samples. Ten pea aphids from each group were evaluated in the assays.

One hundred fly heads were harvested and immediately frozen in liquid nitrogen. Tissues were homogenized using the homogenizer (Precellys 24, Bertin Technologies, Germany) with 300 μl ddH₂O. Then frozen at −80 °C for 30 min and thawed at room temperature, followed by 5 cycles of 30 s on and 30 s off sonication. Lysates were centrifuged using 10,000 g at 4 °C for 5 min. Supernatant was transferred to 10 kDa molecular weight cutoff spin filter (Millipore, USA), followed by centrifugation using 10,000 g at 4 °C for 30 min to remove proteins. Assays were assembled according to the instruction of manufacturer (S0053, Beyotime Biotechnology, China). Absorbance at 412 nm were detected by the Multiskan GO (Thermo Fisher Scientific, USA).