Western blotting detection kit

Human lung squamous carcinoma cell line SK-MES-1 was purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Isoalantolactone was purchased from the Chinese materials research center (Beijing, China), dissolved in Dimethyl Sulfoxide (DMSO), which was purchased from Shenggong Company (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), Hoechst 33342, Dulbecco's Modified Eagle's Medium (DMEM) and Rhodamine 123 mitochondrial specific fluorescent dye were purchased from Sigma (St. Louis, MO, USA). Cell Cycle Analysis Kit (PI+Rnase A), Annexin V-FITC Apoptosis Detection Kit, Reactive Oxygen Species Assay Kit and BCA Protein Assay Kit were purchased from Keygene Company (Nanjing, China). Polyclonal antibodies against β-actin(1:2000 dilution #4967S), Bax(1:1000 dilution #2772S), Bcl-2(1:1000 dilution #2876S), pro-caspase-3(1:1000 dilution #9662P), poly AIsoalantolactone-ribose polymerase (PARP) (1:1000 dilution #9542S), pRb(1:1000 dilution #9306S), p27(1:1000 dilution #2552), p53(1:1000 dilution #9282S), and horseradish peroxidase-conjugated secondary antibodies (goat-anti rabbit, mouse) were purchased from Cell Signaling Technology, Inc. (Shanghai, China). Western Blotting detection kit was purchased from Milipore (Billerica, MA, USA).

We stored heart tissues at −80°C. Proteins from tissues or cells were isolated and extracted using RIPA Buffer (89,900, Roche, Germany) with protease inhibitor (Roche, Germany). A BCA protein assay kit (P0010, Beyotime, China) was used to detect the protein concentration. Protein samples were separated in 8% denaturing sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, United States). Membranes were blocked with skimmed milk powder (Bio-Rad, United States) for 1.5 h and incubated at 4°C overnight with primary antibodies against PPARγ2 (1:1,000, ab45036, Abcam), DDK (1:2,000, TA50011, OriGene), and GAPDH (1:500, TA505454, ZSGB-Bio). Then, the membranes were washed in TBST 3 times, followed by incubation in secondary antibody (horseradish peroxidase-labeled lgG anti-goat/rabbit antibody; 1:2,000, OriGene, China) for 1 h at room temperature. After washing membranes in TBST 3 times, the western blotting detection kit (Millipore, United States) was used for exposure. Densitometric measurements of the bands were performed using FusionCapt Advance software (VILBER, French).

Western blot samples were lysed within RIPA buffer (Pierce, Rockford, IL, USA) containing protease inhibitors. Then, the same amount of proteins was resolved by SDS-PAGE and PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk for one hour, the blots were incubated with primary antibody GATA4 (Abcam, Cambridge, UK) overnight and then with horseradish peroxidase-conjugated secondary antibody for another one hour. Finally, protein bands were visualized using the Western blotting detection kit (Millipore, Bedford, MA, USA) and quantified with Image Pro Plus version 6 software (Media Cybernetics, Rockville, MD, USA).