Sc 398394

The left ventricular specimens for microscopy were fixed in 4% paraformaldehyde, embedded in paraffin, and 2.5 µm-thick sections were prepared. Hematoxylin–eosin (H&E) and Elastica-Masson-Goldner (EMG) staining were performed for histological analysis. Immunohistochemical staining of PPARα and ATF5 were conducted. Briefly, sections were deparaffinized in xylene, rehydrated with a series of graded ethanol, and heated in 10 mM citrate buffer (pH 6.0) for antigen retrieval. 3% hydrogen peroxide was applied afterward to quench the activity of endogenous peroxidase. The sections were then blocked with 0.5% bovine serum albumin serum in PBS. Subsequently, the sections were incubated with rabbit anti-ATF5 monoclonal antibody (diluted 100-fold; ab184923, Abcam, Cambridge, UK) or mouse anti-PPARα monoclonal antibody (diluted 100-fold; sc-398394, Santa Cruz Biotechnology, Inc., USA) overnight at 4 °C, followed by visualization of antigens by use of Histofine simple stain MAX-PO (Multi) (Nichirei Biosciences Inc., Tokyo, Japan) using diaminobenzidine (Nichirei Biosciences Inc.) as a substrate. Sections incubated in PBS in place of the primary antibodies were used as controls for immunostaining procedures.

The mice's left atrium was homogenized in RIPA lysis buffer (P0013 B, Beyotime Biotechnology, China) containing protease inhibitor cocktail (1 : 100, Sigma). Total protein (50 μg) was separated by 10% SDS-PAGE gels and then separated protein was transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk and subjected to primary antibodies overnight at 4°C against ANGPTL4 (1 : 200; ab196746, Abcam), PPARα (1 : 200; sc-398394, Santa Cruz), PPARγ (1 : 200; sc-7273, Santa Cruz); CPT-1 (1 : 500; ab234111, Abcam), SIRT3 (1 : 200; ab246522, Abcam), and ß-actin (1 : 1000; ab8226, Abcam), followed by incubation with HRP-conjugated secondary antibody. Protein bands were visualized using Enhanced Chemiluminescence Plus (Millipore, MA, USA). Relative expression of proteins was normalized to ß-actin.

Total protein was extracted from liver tissues and quantified by the BCA method. Fifty micrograms of protein was loaded onto a 10% SDS-PAGE gel for electrophoresis. The membranes were routinely washed and blocked with 5% nonfat dry milk in PBST (containing 0.05% Tween 20), oscillated at room temperature for 1 h, and incubated overnight at 4 °C with β3-AR (Santa Cruz, sc-515763, 1:500 dilution), peroxisome proliferator-activated receptor-alpha (PPAR-α) (Santa Cruz, sc-398,394, 1:100 dilution), peroxisome proliferator-activated receptor-gamma (PPAR-γ) (Santa Cruz, sc-81,152, 1:500 dilution), mitochondrial carnitine palmitoyltransferase-1 (mCPT-1) (Abcam, Cambridge, ab104662, 1:100 dilution) and CD36 (Santa Cruz, SC-7309, 1:100 dilution) primary antibodies. After rinsing in TBS, the membranes were incubated with secondary antibodies and oscillated at room temperature for 1 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were measured as an internal control with anti-GAPDH (1:1000) antibodies (Zhongshan Golden Bridge Bio, China). A Bio-Rad imaging system and ImageJ software were used to detect the immunoreactive bands and to quantify each sample.

We used the following reagents to detect proteins: monoclonal anti-G3BP (Sigma-Aldrich WH0010146M1), polyclonal anti-TIA1 produced in rabbit (Sigma-Aldrich SAB4301803), anti-GAPDH (sc-47724, Santa Cruz Biotechnology), anti-PPARA (sc-398394, Santa Cruz Biotechnology), anti-PPARG (sc-7273X, Santa Cruz Biotechnology), anti-PPARD (sc-74517, Santa Cruz Biotechnology), antiphospho-4EBP (Ser65, sc-293124), and anti-4EBP (sc-9977, Santa Cruz Biotechnology).
Secondary antibodies for immunofluorescence: anti-rabbit IgG Cy3-conjugated (Sigma-Aldrich C2306), anti-mouse IgG Cy3 conjugated (Sigma-Aldrich C2181), and anti-rabbit IgG Cy5 conjugated (Invitrogen A10523).

M064 cells were lysed in the IP lysis buffer (P0013; Beyotime Biotechnology) according to the provided instructions. Cell lysate containing 200 μg of protein was incubated with the Dynabeads^® Protein G and 2 μg AQP4 antibody (at a dilution ratio of 1: 100, sc‐32739, Santa Cruz Biotechnology) or IgG (negative control) at 4°C for 4 h. Finally, the immunoprecipitation complex was analysed by Western blot using the PPAR‐α antibody (at a dilution ratio of 1:100, SC‐398394, Santa Cruz Biotechnology).