β actin

Total protein from 1 × 10⁶ cells was extracted, and a standard western blot procedure was performed. Briefly, after protein quantification with a BCA protein assay kit (Thermo, Waltham, MA), the protein samples (20 µg) were electrophoresed on 10% SDS polyacrylamide gels and transferred to a PVDF membrane (Millipore, Boston, MA). After being blocked with TBST buffer containing 5% nonfat dry milk, the membranes were incubated with one of the following primary antibodies: Hnf4α (Cell Signaling Technology, Danvers, MA, 1:1000), albumin (Alb, R&D Systems, Minneapolis, MN, 1:1000), E-cadherin (Cdh1, Abcam, Cambridge, UK, 1:1000), Foxa3 (Abcam, 1:1000), or β-Actin (Abcam, 1:1000). After being washed and incubated with horseradish peroxidase-conjugated anti-rabbit/mouse IgG antibodies (GE Healthcare, Chicago, IL, 1:5000), the proteins were visualized with enhanced chemiluminescence reagents (Millipore). Bands were detected with an automatic chemiluminescence imaging analysis system (Tanon, Shanghai, China).

Total protein was extracted from heart tissues using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.), and 20 μg protein/lane was separated by SDS-PAGE on a 5% stacking gel and 10% separation gel. Separated proteins were subsequently transferred to a PVDF membrane (Millipore, Sigma), which was blocked in Protein Free Rapid Blocking Buffer (1X; cat. no. PS108P, Epizyme; Ipsen Pharma) for 2 h at room temperature. Membranes were incubated with primary antibodies against IL-1β (1:1,000; cat. no. ab234437; Abcam), NLRP3 (1:1,000; cat. no. ab270449; Abcam) and β-actin (1:1,000; cat. no. AF0003; Beyotime Institute of Biotechnology) overnight at 4°C. Following primary antibody incubation, membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:3,000; cat. no. D110058; Sangon Biotech Co., Ltd.) or HRP-conjugated goat anti-mouse IgG antibody (1:3,000; cat. no. D110087; Sangon Biotech Co., Ltd.) for 1 h at room temperature. Membranes were incubated with chemiluminescent HRP substrate (MilliporeSigma) at room temperature for 30 sec. Images were obtained using the Universal Hood II System (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ.

Lysis buffer (PRO-PREP, iNtRON Biotechnology, Seongnam, Republic of Korea) was used to extract total protein from the liver and colon tissues of mice. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The gels were then transferred onto PVDF membranes, blocked with 5% skim milk, and incubated with primary antibodies from Cell Signaling Technology [Dnavers, MA, USA; Carbohydrate-responsive element-binding protein (ChREBP, #58069), CCAAT/enhancer binding protein α (CEBPα, #8178), and glucose-6-phosphate dehydrogenase (G6PD, #12263), and β-actin (#4967)] and Abcam [Cambridge, UK; sterol regulatory element-binding transcription factor 1 (SREBP1, #ab28481), acetyl-CoA carboxylases (ACC, #ab45174), fatty acid synthetase (FAS, #ab22759), and Zonula Occludens-1 (ZO-1, #ab214228)] overnight at 4 °C. Next, the membranes were incubated with secondary antibodies. Bands were subsequently quantified using Image Lab Software (Bio-Rad) and an EZ Western Lumi Femto Kit (DoGenBio Co. Ltd., Seoul, Republic of Korea). Finally, chemiluminescence was detected using a ChemiDoc XRS+ imaging system.

To evaluate the effects of acylcarnitine on Akt phosphorylation, H9C2 cells were seeded in 6‐well plates at a density of 1 × 10⁵ cells/mL in a total volume of 2 mL per well. Upon reaching 80% confluence, the cells were treated with 10 μM cis‐OC, trans‐OC, or PC, and DHA or EPA was added at a concentration of 40 μM for 1 h. Basal and insulin‐stimulated (10 nM insulin was added 15 min before the end of incubation) Akt phosphorylation was measured via western blot analysis. The cells were lysed with an ultrasonic processor (Cole‐Parmer instruments, USA) at 20% amplitude for 20 s in lysis buffer at a ratio of 1:10 (w/v) at 4°C. Lysis buffer containing 100 mM Tris–HCl, pH 7.4, 10 mM EDTA, 5 mM MgCl₂, 1 μM DTT, 2% Igepal, and protease inhibitors (10 μM leupeptin, 1 μM pepstatin, 1 μM aprotinin, and 100 μM PMSF) was used. PAGE and western blot analysis of the tissue lysates were performed as described previously.
²⁵ (link) To measure the level of phosphorylated Akt at Ser473, the membranes were incubated with anti‐P‐Akt (#sc‐7985‐R; Santa Cruz Biotechnology, CA, USA or #4060S; Cell Signaling Technology, Danvers, MA, USA) antibodies, and the values were normalized to the protein level of beta‐actin (Abcam, ab8224). The blots were developed via the use of chemiluminescence reagents (Millipore), and images were captured using an Azure c400 gel imaging system.