Protein phosphatase inhibitor cocktail

To extract protein from HUVECs, a protein extraction kit containing protease inhibitors and a protein phosphatase inhibitor cocktail was used (Thermo Fisher Scientific). Equal amounts of protein (30 µg/lane) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Blots were probed overnight at 4°C with primary antibodies (1:1,000), washed with Tris-buffered saline containing Tween 20, and incubated with secondary antibodies (1:10,000; ZSGB-BIO, Beijing, China) for 1 h at room temperature. Finally, blots were washed, incubated with SuperSignal™ WestFemto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and analyzed using a ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, United States).

Cells or tissues were lysed in RIPA buffer, including a protein-phosphatase inhibitor cocktail (Thermo Scientific, USA) at 4℃, then use 10% SDS-PAGE gels to separate the total proteins before they are transferred electrophoretically (Bio-Rad, USA) onto PVDF membranes (Millipore, USA). Bio-Rad ChemiDoc XRS +System was used to detect the protein after dropping the developer solution (Bio-Rad, USA) onto the blots. The primary antibodies were purchased in this study as follows: anti-LAMC1 (PAB32791; 1:1000; Bioswamp, China), anti- CEBPα (18311-1-AP; 1:1000; Proteintech, USA), anti- PPARγ (16643-1-AP; 1:1000; Proteintech, USA), anti-DLK1 (10636-1-AP; 1:1000; Proteintech, USA), anti-HSL (17333-1-AP; 1:1000; Proteintech, USA), anti-Perilipin-1 (9349; 1:1000; Cell Signaling, USA), anti-MEK1/2 (11049-1-AP; 1:1000; Proteintech, USA), anti-p-MEK1/2 (phosphor Ser217/221) (3958; 1:1000; Cell Signaling, USA), anti-ERK1/2 (16433-1-AP; 1:1000; Proteintech, USA), anti-p-ERK1/2 (phosphor Thr202/Tyr204) (AF1015; 1:1000; Affinity Biosciences, USA), anti-STAT3 (10253-2-AP; 1:1000; Proteintech, USA), anti-p-STAT3 (phosphor Tyr705) (9145; 1:1000; Cell Signaling, USA), mTOR (2983T; 1:1000; Cell Signaling, USA), anti-p-mTOR (phosphor Ser2448) (5536T; 2983T; 1:1000; Cell Signaling, USA), anti-GAPDH (sc-32233; 1:5000; Santa Cruz, CA), and anti-β-tubulin (sc-5274; Santa Cruz, CA).

Treated cells were washed with PBS twice and then harvested using ice-cold RIPA lysis buffer containing protease inhibitor PMSF (Gibco) and protein phosphatase inhibitor cocktail (Gibco). The lysates were centrifuged at 12,500 g for 20 min at 4°C and the supernatant fractions were collected. Protein concentrations were measured with BCA Protein Assay Kit (Gibco). After denaturation at 95°C for 10 min, equivalent aliquots of protein samples (30 μg) were loaded and electrophoresed on SDS-PAGE gels and then transferred to PVDF membrane (Thermo Scientific). The membranes were firstly blocked with 5% nonfat dry milk for 2 h at room temperature and then incubated with primary antibodies (1 : 3000) overnight at 4°C. Then HRP-linked secondary antibodies (1 : 5000) were incubated for 4 h at room temperature. The bands were visualized with the ChemiDoc™ MP Imaging System (Bio-Rad).