Odyssey infrared fluorescent scanner

Caco-2 cells were cultured in MEM and then treated with test samples for indicated time. Proteins were isolated by lysis buffer (Beyotime, China) and measured using the Nanodrop 1000 Spectrophotometer (Thermo, USA). Protein samples were separated on 10% SDS-polyacrylamide gels (SDS-PAGE) and transferred onto the PVDF membranes (Millipore, USA). After blocked with 1% BSA in TBST for 2 h, membranes were incubated with primary antibodies overnight at 4°C. Blots were washed and incubated with secondary antibodies for 1 h at room temperature. Membranes were again washed three times with TBST and were scanned with an Odyssey infrared fluorescent scanner (LI-COR) and analyzed with Odyssey software version 3.

Seed PC3 cells in six-well plates at a density of 5 × 10⁵ cells per well, in a CO₂ incubator overnight and treated with QLXZD (0–30 mg/mL) for 24 h. The total protein were collected as previously described (Liu et al., 2017 (link)). The equalized amounts of proteins from each sample were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to polyvinylidene fluoride (PVDF) membranes. After blocking with 1% (w/v) albumin from bovine serum (BSA) for 2 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by IRDye-conjugated secondary antibodies for 1 h at room temperature and scanned with an Odyssey infrared fluorescent scanner (LI-COR Biosciences). GAPDH was used as the endogenous control.

For protein electrophoresis and immunoblotting, the cerebral cortex and striatum were homogenized on ice with lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protease and phosphatase inhibitor min-tablets (Thermo Scientific, USA) were added according to the manufacturer’s instructions. After centrifugation, protein extracts were collected, and 50 μg of protein lysates were resolved by SDS-PAGE and electrophoretically transferred onto Immobilon-P membranes (0.22 μM; Millipore). Membranes were blocked with 5% non-fat milk for 2 h, then incubated with primary antibodies overnight at 4 °C. After washing with PBS-T, membranes were incubated with the fluorescent-conjugated secondary antibodies in PBS-T/milk for 2 h at room temperature. Membranes were then rewashed, after which the labelled proteins were detected using an Odyssey infrared fluorescent scanner (LI-COR) with Odyssey ver. 3 software used for analysis.

Abdominal aorta and myocardial proteins were extracted according to the method provided by the manufacturer of the extraction kit (Key Gen Bio TECH, Nanjing, China).The concentrations of total protein in the supernatant were determined by the BCA method, and then 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol were added to denature proteins at 100°C for 10 min. Fifty micrograms of protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with 5% non-fat milk in 20 mmol/L Tris-buffered saline (pH 7.4) containing 500 mmol/L NaCl and 0.05% Tween 20 (TBST) and then incubated with primary antibody (1:150 dilution, PPAR-α, SC-1982; PPAR-γ, SC-1981; and GAPDH, SC-166545: Santa Cruz Biotechnology, USA; PPAR-β/δ, ab23673: Abcam, USA) overnight at 4°C. Thereafter, the membranes were washed three times with TBST and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Membranes were again washed three times in TBST. Finally, signals were detected using the Odyssey Infrared Fluorescent Scanner (LI-COR, USA) and quantified by Image J software.