Complete cocktail of protease inhibitors

Proteins were extracted using the PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) supplemented with a complete cocktail of protease inhibitors (Roche, Basel, Switzerland) for 15 min on ice. After centrifugation at 14,000 g for 15 min at 4℃, the supernatant was collected, and equal volumes of protein from each sample were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were blocked in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TBST) and 5% skim milk. After being washed once in TBST, membranes were incubated overnight at 4℃ with the primary antibodies diluted in TBST supplemented with 5% BSA. The antibodies used included rabbit polyclonal anti-mTOR, rabbit polyclonal anti-AKT, rabbit polyclonal anti-p70S6K (all from Cell Signaling Technology Inc., Beverly, Massachusetts, USA), and mouse monoclonal anti-α tubulin (Santa Cruz Biotechnology, Santa Cruz, California, USA). Following 3 consecutive washes in TBST, membranes were incubated for 90 min with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) diluted 1:10,000 with incubation buffer, as described above. After extensive washing, bound secondary antibodies were visualized using an enhanced ECL chemiluminescence detection kit (GE Healthcare, Little Chalfont, UK).

Mitochondrial and cytosolic fractions of FHs 74 Int cells were prepared as described previously [PMID: 32547023]. For cytosolic extracts free of nuclei and mitochondria, the cells were washed in ice-cold PBS (pH 7.2) and then in hypotonic extraction buffer (HEB; 50 mM PIPES, 50 mM KCl, 5 mM EGTA, 2 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4) and harvested by centrifugation. Pellets were resuspended in HEB and lysed with a Dounce homogenizer. These cell lysates were then centrifuged at 100,000×g for 60 min at 4°C, and the supernatants were flash-frozen in cold ethanol, aliquoted, and stored at −80°C. Mitochondrial fractions were prepared by washing the cells in ice-cold PBS and then resuspending them in an isotonic homogenization buffer (10 mM Tris-HCl, pH 7.5, 250 mM sucrose, 10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, EDTA-free complete cocktail of protease inhibitors [Roche]). After 60 strokes in a Dounce homogenizer, unbroken cells were removed by centrifugation at 1,000×g for 10 min, and the supernatants were centrifuged at 14,000×g for 20 min. The supernatants were collected as the mitochondrial fraction. The expression of cytochrome c expression was determined by western blotting. COX IV and α-tubulin were used as mitochondria and cytosol markers, respectively.

bEnd.3 cells were homogenized in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.57% sodium deoxycholate, 0.1% SDS, 50 mM Tris [pH 8], 1× Complete cocktail of protease inhibitors; Roche, Basel, Switzerland). Homogenates were centrifuged at 13,000 rpm for 10 min. Supernatants were mixed with 4× Laemmli sample buffer (200 mM Tris/HCl [pH 6.8], 8% SDS, 40% glycerol, 50 mM EDTA, 0.08% bromophenol blue) and 1% dithiothreitol. Lysates were heated at 95°C for 10 min before performing standard SDS-PAGE and western blotting. The following primary antibodies were used: rabbit anti-GAPDH (1:20,000 in 5% BSA; 2118, Cell Signaling Technology), rabbit anti-GluN1 (1:2,000 in 5% milk; 5704, Cell Signaling Technology), rabbit-GluN2A (1:500 in 5% milk; AB1555, Merck Millipore), rabbit anti-GluN2B (1:1,000 in 5% milk; NB300-106, Novus Biologicals, Centennial, CO, USA), rabbit anti-GluN2C (1:1,000 in 5% milk; PAB9705, Abnova, Taiwan, China), and rabbit anti-GluN2D (1:1,000 in 5% milk; AP21181PU-N, Acris OriGene, Herford, Germany). The following secondary antibody was used: peroxidase AffiniPure goat anti-rabbit IgG (H+L) (1:5,000 in 5% milk; 111-035-144, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Membranes were developed with enhanced chemiluminescence (ECL) solution (GE Healthcare, Chicago, IL, USA) and the ChemiDoc imaging system (Bio-Rad).

The effect of PF2 on α-amylase activity of
Z. subfasciatuslarvae was determined using the Bernfeld method (
Noelting and Bernfeld 1948 (link)
). The crude extract (2 µl) of midgut from 20-d-old larvae with a complete cocktail of protease inhibitors (Roche), was incubated with PF2 (100 µg/ml) in 100 mM acetate buffer, 20 mM NaCl, and 0.2 mM CaCl
₂, pH 6.8 at 30°C. After 3 min of incubation with PF2, 25 µl of 1% soluble starch was added and incubated at 30°C for another 15 min. The assay was stopped with the addition of 100 µl of 3,5-dinitrosaliciylic acid, heated in boiling water for 10 min, cooled and diluted with 1 ml of water, and the absorbance was measured at 540 nm. The α-amylase activity was expressed in milligrams of maltose liberated/10 min/37°C. The α-amylase inhibitory activity was expressed as a relative α-amylase activity without preincubation with PF2. Assays were completed in triplicate. Maltose was used as the standard with one unit of enzyme activity defined as the amount of enzyme required to produce 1 μM of maltose/min.

Whole proteins of exponentially growing cells or tissues were collected in lysis buffer (Promega) containing the complete cocktail of protease inhibitors (Roche, Branchburg, NJ, USA). The protein concentration of the cell lysates was determined by Bradford methods (Beyotime) and then was regulated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Equal amounts of total proteins were loaded to 12% polyacrylamide gels and electrophoresed to separate proteins of interest at 120 V for 2 h, then the proteins were transferred to PVDF membranes (Millipore Corporation, Bedford, MA, USA) using a semi-dry method (Bio-Rad, Hercules, CA, USA). The membranes were blocked and incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 h at 37°C. At last, targeted proteins were detected through the ECL protocol (Beyotime), scanned and analyzed by automatic image analyzer. β-actin was used as a control and obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All of the primary antibodies and dilutions contained the following: donkey anti-SNF1 antibody (1:2000; Santa Cruz), mouse anti-HSF1 antibody (1:1000; Santa Cruz), rabbit anti-p53 antibody (1:500; Cell Signalling, Danvers, MA, USA), and rabbit anti-EGFP antibody (1:500; Beyotime). Secondary antibodies coupled to horseradish peroxidase (HRP) were purchased from Beyotime.