Protein assay kit

Individual (for linkage experiments) or pooled (for binding assays) guts dissected from fourth instar S. frugiperda larvae were used for preparation of BBMV by the differential centrifugation method^{33 (link)} as previously modified^{34 (link)}. The final BBMV pellet was resuspended in ice-cold PBS solution (2 mM KCl,135 mM NaCl, 1.7 mM KH₂PO₄,10 mM Na₂HPO₄, pH 7.5) and total protein quantified using the Qubit fluorimeter using a protein assay kit (Invitrogen). The purity of the final BBMV preparations was determined using the enrichment in aminopeptidase-N (APN) and alkaline-phosphatase (ALP) activities compared to initial midgut homogenates as described elsewhere^{35 (link)}. The APN and ALP activities were enriched 4–5 times in the final BBMV preparation compared to the initial midgut homogenates.

Histone proteins were isolated using Histone Extraction Kit (Abcam), following the manufacturer’s instructions on days 0 and 7 of adipogenesis in three biological replications. Briefly, cells were detached from plates by trypsinization in 0.25% Tripsin-EDTA solution (Sigma) for 5 min at 37 °C, in line with the standard procedure. Cells were then resuspended in 1 × Pre-Lysis Buffer at a concentration of 10⁷ cells/ml and lysed for 10 min with gentle stirring at 4 °C. The cells were then collected by centrifugation at 10,000g for 1 min at 4 °C. Cells were resuspended in Lysis Buffer (200 µl per initial 10⁷ cells) and incubated at 4 °C for 30 min. The cell suspension was then centrifuged at 12,000g for 5 min at 4 °C, and the supernatant, containing acid-soluble proteins, was transferred to a new vial. Finally, 3 µl the Balance-DTT Buffer was added for each 10 µl of supernatant. Protein concentration was measured using Qubit 2.0 Fluorometer (Invitrogen) with Protein assay kit (Invitrogen).

The quantitative protein method is one of the common quantitative methods of exosomes. In this paper, the concentration of exosomes was measured by a fluorometer (Qubit 3.0, Waltham, MA, USA) and a protein assay kit (Invitrogen, Waltham, MA, USA). The standard curve was set up firstly using different concentrations (0, 200, 400 μg/mL) of standard protein samples. The concentration of exosomes was quantified using the relative protein content. The capture efficiency was calculated by the following equation: $$\eta =\frac{{\eta }_0-{\eta }_1}{{\eta }_0}$$
where $${\eta }_0$$ denotes the relative concentration of exosomes of original samples, $${\eta }_1$$ denotes the relative concentration of exosome after capture.

EPA and AA, dimethyl sulfoxide (DMSO), 3-(4, 5-dimethyl-thiazol-2-y)-2.5-diphenyl tetrazolium bromide (MTT), Phorbol-12-myristate-13-acetate(PMA) and Oil Red O were obtained from Sigma–Aldrich USA. Phosphate buffered saline (PBS) tablets were provided by Takara, Japan. Fetal bovine serum (FBS) were obtained from Invitrogen Corporation. Ox-LDL was purchased from YiYuan Biotech (Guangzhou, China). Fatty acids free BSA was obtained from Equitech-bio company (US). The ELISA kits of IL-6 and TNFα was purchased from Science Biotechnology Co. Ltd. (Yantai, China). The cholesterol assay kit was purchased from Applygen Technologies Inc. (Beijing, China). The protein assay kit was purchased from Thermo Fisher Scientific (Rockford, IL). Reagents and kits for RNA extraction and reverse transcription were obtained from Qiagen, USA.

RIPA (Radio Immunoprecipitation Assay) Lysis and Extraction Buffer (Thermo Scientific ™) was added to frozen liver tissue samples, and they were grinded in a homogenizer. The obtained tissue homogenate was centrifuged at 15,000 rpm (10 min, 4°C) and the supernatant was left. Then the Pierce ™ BCA (bicinchoninic acid) Protein Assay Kit (Thermo Scientific ™) was used to determine protein concentration. Equal amounts of proteins were separated by 10–12% SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skim milk for 3 h on a shaker at room temperature and then incubated with the primary antibodies (diluted with 5% skim milk) overnight at 4°C, and the concentrations of the primary antibodies are as follows: the concentration of primary antibody used for GAPDH, p-AKT, AKT, Nrf2, HO-1, NLRP3, p-PI3K, PI3K, and Caspase-1 was 0.5 μg/ml, and the concentration of primary antibody used for PPARγ, and Caspase-3 was 1 μg/ml. The membranes were washed via Tris-Buffered Saline Tween-20 (TBST) three times for 10 min each time and then incubated with the secondary antibodies (diluted with TBST) on the second day. Finally, the membranes were visualized with the chemiluminescent HRP substrate after washed again by TBST and analyzed via Image J gel analysis software.

Cells were lysed in Lysis Buffer (Hepes 10 mM, NaCl 150 mM, SDS 1%, EDTA 4 mM, protease and phosphatase inhibitors). Protein concentration was quantified using BCA assay (Protein Assay Kit, Thermo Fisher). Ten micrograms of total cell lysates were loaded into NuPage 4–12% Bis-Tris gels and separated by electrophoresis in MES buffer (Life Technologies, B0002). For Western blotting, proteins were transferred onto Protran nitrocellulose membranes and saturated for 1 h in PBS-T (PBS, 0.1% Tween 20) supplemented with 5% BSA (Sigma, A4503-100G). Incubation with primary antibodies was performed o.n. at 4 °C. Membranes were washed and incubated with specific HRP-conjugated secondary antibodies for 1 h. After additional washings, signals were revealed with Luminata^TM using an Uvitec gel doc system (Uvitec, Cambridge, UK).