Ab3484

The antibody against PPARα (peroxisome proliferator-activated receptor alpha, 66836-1-Ig) was purchased from Proteintech Co. (Chicago, USA). The antibodies against SREBP1 (sterol regulator element-binding protein 1, ab28481) and P-PPARα (phosphor-PPARα, S12, ab3484) were purchased from Abcam Co. (Cambridge, UK). The antibody against GAPDH (2118S) was purchased from Cell Signaling Technology Co. (MA, USA). The animal treatment and experimental procedures are carried out according to the “Guidelines for the Care and Use of Experimental Animals” issued by the National Research Council. All animal care and use procedures have been approved by the Animal Ethics and Welfare Committee of Guangdong Ocean University (license number: DOU-AEWC-20180063), and all authors have clearly stated that they have followed these guidelines.

Huh-7 or HepG2 cells were collected in radioimmunoprecipitation assay (RIPA) (Beyotime Biotechnology, Shanghai, China) lysis buffer containing 1% phenylmethylsulphonyl fluoride (PMSF) (Beyotime Biotechnology, Shanghai, China) on ice for 10 min. The cell lysates were removed and placed into 1.5 ml Eppendorf tubes and centrifuged at 12,000 rpm for 15 min. Next, the supernatants were used to determine the total protein concentration using the bicinchoninic acid (BCA) (Beyotime Biotechnology, Shanghai, China) assay. Protein samples (25 μg) were loaded into each well and separated with 5% concentrated gels and 12% separation gels. After blocking the membranes with 5% defatted milk, rabbit anti-human FABP1 (1 : 1000, SAB1410361, Sigma-Aldrich), mouse anti-human caspase 3 (1 : 1000, MAB10753 Sigma-Aldrich), mouse anti-human CPT1A (1 : 1000, ab128568, ABCAm), rabbit anti-human PPAR-α (phospho S12, 1 : 1000, ab3484, ABCAm), and rabbit anti-human SREBP1 (1 : 1000, ab191857, ABCAm) antibodies were added and incubated at 4°C overnight. Next, the membranes were incubated with goat anti-rabbit (12348, Sigma-Aldrich) or goat anti-mouse (12349, Sigma-Aldrich) secondary antibody (1 : 5000) for 2 h at room temperature and developed with enhanced chemiluminescence (ECL) kit (Millipore, Burlington, MA, USA). The gels were imaged using the Syngene gel imager (Frederick, MD, USA).

The frozen placenta was thawed, washed with radioimmunoprecipitation assay buffer (p0013b, Beyotime, Beijing, China), and cut into 2–3 mm pieces, followed by homogenization and ultrasonication. Then, the supernatant was used for the quantification of protein concentration using bicinchoninic acid assay (p0010s, Beyotime, Beijing, China). The protein samples were separated with 8–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, washed with TBST buffer thrice for 5 min each time, and then blocked with 5% skimmed milk for 2 h. The samples were then incubated with primary antibodies against PPARγ (CST, #2443, 53 kDa, 1:1000), PPARα (ab3484, 52 kDa, 1:1000; Abcam, Cambridge, UK), ANGPT1 (ab8451, 57 kDa, 1:500; Abcam, Cambridge, UK), VEGF (CY2367, 28 kDa, 1:1000; Abways, Kärnten, Austria), β-actin (AB0033, 42 kDa, 1:5000; Abways, Kärnten, Austria), HIF1α (#36169, 120 kDa, 1:1000; CST, Danvers, MA, USA), PIGF/PIGF (ab196666, 25 kDa, 1:500; Abcam, Cambridge, UK), TNFα (#3707, 17 kDa, 1:1000; CST, Danvers, MA, USA) at 4 °C for 10 h. After washing thrice, the samples were incubated with secondary antibody (1:10000; Abways, Kärnten, Austria) at room temperature for 1 h. The bands were captured using enhanced chemiluminescence detection reagent and Tanon imaging system (Tanon-5200Multi, Shanghai, China).

The protein concentration of ventricular extracts was measured using the bicinchoninic acid assay (BCA1, Sigma-Aldrich, USA). 30 μg of total extract protein from rat ventricular extracts were separated by SDS-PAGE and then wet transferred onto nitrocellulose membranes (HATF00010, Millipore, USA). Membranes were immunoblotted with primary antibodies against UCP1 (ab10983, Abcam, USA), AMPK α1 + AMPK α2 (ab80039, Abcam, USA), phosphor-AMPK α1 (T183) + AMPK α2 (T172) (ab133448, Abcam, USA), mTOR (2972S, Cell Signaling Technology, USA) and phosphor-mTOR (S2448) (2971, Cell Signaling Technology, USA), PPARα (ab24509, Abcam, USA) and phosphor-PPARα (S12) (ab3484, Abcam, USA). Protein bands were detected using horseradish peroxidase-conjugated secondary antibodies using chemiluminescent detection reagent (sc-2048, Santa Cruz, USA). GAPDH (ab9482, Abcam, USA) was used as normalization. Bands were quantitatively analyzed with ImageJ software.