Rabbit anti plin2

Oxymatrine (purity >98%) was purchased from Sigma-Aldrich. Hepatic TG and FFA assay kits were from Nanjing Jiancheng Bioengineering Institute. Sequencing-grade trypsin was from Promega. iTRAQ reagent-8 plex multiplex kit was obtained from Applied Biosystems. Triple TOF 5600 mass spectrometer and Eksigent nanoLC-1D plus liquid chromatography were from SCIEX. Rabbit anti-Plin2, rabbit anti-L-FABP, rabbit anti-FASN, rabbit anti-Sirt1 and mouse anti-SCD1 monoclonal antibodies were from Abcam. Rabbit anti-AMPKα (phospho-Thr172) and rabbit anti-AMPKα monoclonal antibodies were from Cell Signaling Technology.

ISCs were divided into treated and control groups, with PA-mixed medium (300 μM) or with 0.05% BSA for 48, 72, and 96 h, respectively. At different in vitro culturing times, experiments were performed using the standard protocol [19 (link)] with the primary antibodies specific for the following proteins: rabbit anti-PLIN2 (Cat#ab108323, Abcam, UK), rabbit anti-PLIN3 (Cat#ab47638, Abcam, UK), Col I (Cat#ab34710, Abcam, UK), rabbit anti-FN (Cat#ab2413, Abcam, UK), rabbit anti-perilipin 4 (PLIN4) (Cat#10694-1-AP, Proteintech, USA), mouse anti-PLIN5 (Cat#sc-514296, Santa Cruz, USA), mouse anti-β-actin (Cat#TA811000, Origene, China), mouse anti-α-SMA (Cat#A2547, 1 : 3000, Sigma, USA), rabbit anti-P-Smad3 (Cat#9520, CST, USA), rabbit anti-Smad3 (Cat# 8685S, CST, USA), and rabbit anti-TGF-β (Cat#3711, CST, USA). Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit (Cat#SE134, Solarbio, China) or anti-mouse (Cat#SE131, Solarbio, China) antibody was used as the secondary antibody. Quantitative analysis of proteins was performed using enhanced chemiluminescence (Millipore, USA) and Image J software (National Institutes of Health, MD, USA), respectively.

Cell proteins were extracted with a protein extraction Kit (Solarbio Company, Beijing, China), then mixed with protein loading buffer and denatured at 100 °C for 10 min with 1 mM phenylmethylsulfonyl fluoride (Millipore, Billerica, MA, USA), and 1 mM ethylenediaminetetraacetic acid (EDTA). The membranes were then incubated sequentially with antibodies against GAPDH (rabbit anti-GAPDH, 1:10,000 Abcam, Cambridge, UK) and PLIN2 (rabbit anti-PLIN2, 1:800 Abcam NT, HK) for 12 h at 4 °C. After washing three times (10 min each) with PBS-Tween 20, the membranes were incubated with secondary IgG-Goat anti-Rabbit HRP antibodies (1:2000 NOVUS NT, HK) diluted in PBS-Tween 20 (0.08 μg/mL) for 2 h, and then washed in PBS-Tween 20. Chemiluminescent detection (Millipore) was performed by mixing equal volumes of Luminol Reagent and Peroxide solution in a clean container or test tube and then applying the mixture to PVDF membranes. Immunoreactivity was detected using a Gel Doc™ XR+ Gel Documentation System (Bio-Rad, Hercules, CA, USA).