Native page 4 to 16 mini protein gel

THP1 cells (8.8 × 10⁶ cells) differentiated into macrophages were replaced with serum-free medium for 16 hours, washed twice with PBS, and then after the addition of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), the cells were scraped and centrifuged at 800g for 10 minutes at 4 °C. The supernatant was discarded, and the pellet was washed twice with 25 mM HEPES (pH 7.0) by centrifugation at 15 000g for 60 minutes at 4 °C. The pellet was then dissolved in Native PAGE sample buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 0.5% digitonin with or without either FAM-ANGT_HUMAN[448–462] or a fluorescent-labeled peptide of an unrelated sequence (GQGAHHAAGQAGNEAGR, SBSN_HUMAN[243–259]) (10^-4 M) and incubated at room temperature for 30 minutes. The supernatant was collected after centrifugation at 20 000 × g for 30 minutes, and the protein concentration was quantified with a Nano Drop Lite UV-Vis spectrophotometer (Thermo Fisher Scientific), and 20 μg proteins/lane were subjected to clear-native polyacrylamide gel electrophoresis (CN-PAGE) using Native PAGE 4% to 16% Mini Protein Gel (Thermo Fisher Scientific) at 120 V for 120 minutes. Fluorescence was detected using a light-emitting diode (LED) illuminator. Native Mark Unstained Protein Standard (Thermo Fisher Scientific) was used as the molecular weight marker.

An 8-week-old female Jcl:Wistar rat (CLEA Japan, Tokyo, Japan) was deeply anesthetized with isoflurane, euthanized by decapitation, and the cerebrum was rapidly removed and homogenized on ice using a BL3000 homogenizer (Shinto Scientific Co., Tokyo, Japan) in 10 mL of 50 mM Tris-HCl buffer (pH 7.4) containing protease inhibitor cocktail (Nacalai Tesque Inc.). Homogenates were incubated with 0.5% digitonin (Thermo Fisher Scientific) for 4 hours at 4 °C and centrifuged at 20 000g for 60 minutes at 4 °C. The protein concentration of the supernatant was determined using a Nano Drop Lite UV-Vis spectrophotometer (Thermo Fisher Scientific). Aliquots of the supernatant were diluted with Native PAGE sample buffer (Thermo Fisher Scientific) to 2 μg/μL, incubated with 10^-5 M FAM-ANGT_HUMAN[448–462] for 1 hour at 37 °C, and subjected to CN-PAGE on Native PAGE 4% to 16% mini protein gel (Thermo Fisher Scientific) and Native PAGE running buffer (Thermo Fisher Scientific) for 120 minutes at 120 V. The marker used was Native Mark Unstained Protein Standard (Thermo Fisher Scientific). Fluorescence was visualized using an LED illuminator and photographed, and fluorescent positive bands, together with negative gels of neighboring lanes at the same migration distance, were resected for in-gel trypsin digestion and subsequent LC-MS/MS analysis.