Anti cpt2

For each kidney sample total protein was isolated using RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and global protein was measurement by BCA™ Protein Assay Kit (Pierce, EUA). Around 50 μg of tissue protein was utilized for SDS-PAGE electrophoresis on 12.5% polyacrylamide gels. The immunostaining was performed with primary antibodies (Anti-Cofilin/1:1,000, Abcam, USA, Anti-PPAR-α/1:300, Abcam, Anti-CPT1A/1:1,000, Abcam, Anti-CPT-2/1:500, Abcam and Anti-Beta Actin/1:5,000, Cell Signaling, USA), followed by conjugated secondary antibodies (anti-mouse or anti-rabbit peroxidase/1:250.000, Sigma, USA). After, the nitrocellulose membrane was revealed by chemiluminescence methods using the ECL kit (Millipore, USA), and the images were acquired on Amersham Imager 600 equipment (GE Healthcare, UK). The SyncMaster 740 n software device (GE Healthcare, UK) were used to analyze and quantify the gel bands. The experiment was reapeated twice.

Total proteins were extracted from liver tissue using a protein extraction kit (Minute Protein Extraction Kit; Invent Biotechnologies, Inc., Eden Prairie, MN, USA). Extracted proteins were quantitated by the BCA method (BCA protein Assay Kit; Takara Bio, Inc., Otsu, Japan), and were diluted to the same protein concentrations. Equivalent amounts (50 µg) of each group were run on SDS polyacrylamide gels. Proteins were transferred electrically to a PVDF membrane and incubated with the following antibodies; anti-TNF-α, anti-IL1β (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-DMT1, anti-hepcidine25, anti-CPT1A, anti-CPT2, anti-SOD2, anti-catalase (Abcam, Tokyo, Japan), anti-GPx4 (LifeSpan BioSciences, Seattle, WA, USA). Membranes were washed and incubated with respective secondary antibodies. Detection of bands was performed by chemiluminescent analysis. Beta actin was used as a loading control. Representative data of three experiments were shown on figures.

Total proteins from flight muscles of WT and ACP^-/- locusts were extracted using TRIzol reagent, as previously described (Hou et al., 2019 (link)). The protein extracts (50 μg) were electrophoresed on 4–20% Biofuraw precast gels (Tanon, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was incubated with polyclonal antibody against target protein (anti-FABP, developed by ABclone, China, 1:5000; anti-CPT2, Abcam, 1:1000; anti-ACDM, Abcam, 1:1000). Goat anti-rabbit IgG (EASYBIO, 1:5000) was used as the secondary antibody. Polyclonal antibody against tubulin was used as an internal control. Protein bands were detected by chemiluminescence (ECL kit, Thermo Scientific).