Luminol enhancer solution

3T3-L1 cells were treated with ginkgolide C in 6-well plates, and cells were lysed with protein lysis buffer containing protein inhibitor cocktail (Sigma). Protein doses were calculated using a BCA protein assay kit (Pierce). Equal amounts of the denatured protein were run on 8–10% sodium dodecyl sulfate-polyacrylamide gels, followed by transfer to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dried milk in TBST buffer (150 mM NaCl, 10 mM Tris-HCl pH 8.0, 0.1% Tween 20) for 1 h and then incubated overnight at 4°C with the following respective primary antibodies: phosphorylated-AMPKα, AMPK, fatty acid synthase (FAS), fatty acid-binding protein (aP2), SREBP-1c, and lipoprotein lipase (LPL) (Santa Cruz, CA, USA); phosphorylated acetyl-CoA carboxylase-1 (pACC-1), ACC-1, PPAR-α, PPAR-γ, C/EBPα, C/EBPβ, phosphorylated hormone-sensitive lipase (HSL, pHSL), HSL, and adipose triglyceride lipase (ATGL) (Epitomics, Burlingame, CA, USA); sirtuin 1 (Sirt1) (Millipore); and β-actin (Sigma). The membranes were washed with TBST buffer and then incubated with secondary antibodies for 1 h at room temperature. Finally, membranes were treated with luminol/enhancer solution (Millipore) and exposed using the BioSpectrum 600 system (UVP, Upland, CA, USA).

Lung tissues were homogenized and proteins separated on 10% SDS polyacrylamide gels. The proteins were transferred into polyvinylidene difluoride membranes and incubated with primary antibodies at 4 °C. The next day, the membranes were incubated with secondary antibodies and treated with Luminol/Enhancer Solution (Millipore) to obtain specific protein signals using the BioSpectrum 600 system (UVP, Upland, CA, USA). Primary antibodies included COX-2, HO-1, Nrf2, Lamin B1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and β-actin (Sigma).

Cells were treated with acacetin and lysed using protein lysis buffer (Sigma). Equal amounts of protein were separated on 8–10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, United States). The PVDF membranes were blocked, incubated with primary antibodies overnight at 4°C, incubated with secondary antibodies, and the signal was detected with Luminol/Enhancer solution (Millipore). Protein bands were quantitated using the BioSpectrum 600 system (UVP, Upland, CA, United States). The primary antibodies included antibodies that recognized the following proteins: phosphorylated-AMPKα, AMPK, IκBα, phosphorylated-IκBα, p65, and lamin B1 (Santa Cruz, CA, United States); ERK1/2, phosphorylated-ERK1/2 (pERK1/2), JNK, phosphorylated-JNK (pJNK), p38, phosphorylated-p38 (pp38), fatty acid synthase (FAS), fatty acid binding protein (aP2), sirtuin 1 (Sirt1), SREBP-1c, and lipoprotein lipase (LPL) (Cell Signaling Technology, Danvers, MA, United States); acetyl CoA carboxylase-1 (ACC-1), phosphorylated-ACC-1 (pACC-1), adipose triglyceride lipase (ATGL), C/EBPα, C/EBPβ, PPAR-α, PPAR-γ, hormone-sensitive lipase (HSL), phosphorylated-HSL (pHSL) (Abcam, Cambridge, MA, United States); and β-actin (Sigma).

MDA-MB-231 cells were treated with LA and lysed using RIPA buffer containing protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA). Extracted proteins were separated by 10–15% SDS polyacrylamide gel electrophoresis, and then proteins were transferred from the gel to PVDF membrane. Next, the PVDF membrane was incubated with specific primary antibodies overnight and secondary antibodies at room temperature for 1 h. Proteins were detected using Luminol/Enhancer solution (Millipore, Billerica, MA, USA) and specific proteins demonstrated and measured by the BioSpectrum 600 system (UVP, Upland, CA, USA). Primary antibodies included AKT, p65, PI3K, phosphorylated AKT, phosphorylated p65, phosphorylated PI3K (Santa Cruz, CA, USA), p38, JNK, phosphorylated p38, phosphorylated JNK (Millipore, Billerica, MA, USA), ATG5, Beclin 1, Bcl2, Bax, cleaved caspase-3, cleaved caspase-9, cleaved PARP1, cyclin D1, cytochrome c, E-cadherin, LC3B, p21, p62, γ-H2AX, vimentin (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma, St. Louis, MO, USA).

Protein samples were separated on 10% SDS polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the PVDF membranes were incubated overnight at 4°C with specific primary antibodies against β-actin (Sigma), COX-2, iNOS, HO-1, AKT, pAKT (Santa Cruz, CA, USA), JNK, pJNK, p38, and pp38 (Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were washed three times using tris-buffered saline with Tween 20 (TBST) buffer and incubated with secondary antibodies at room temperature for 1 h. Proteins were detected using Luminol/Enhancer solution (Millipore), and signals were detected using the BioSpectrum 600 system (UVP, Upland, CA, USA) to quantitate protein bands.

Protein extracts were prepared using a protein lysis kit (Sigma) and then separated on 8–10% SDS–PAGE gels. Next, gels were transferred to polyvinylidene difluoride (PVDF) membranes and incubated with primary specific antibodies overnight. The PVDF membrane was washed and incubated with secondary antibodies at room temperature for 1 h. Finally, Luminol/Enhancer solution (Millipore, Billerica, MA, USA) was added to detect specific protein expression using the BioSpectrum 600 system (UVP, Upland, CA, USA). Primary specific antibodies included β-actin (Sigma), ATGL, HSL, phosphorylated HSL (pHSL), C/EBPα, C/EBPβ, PPAR-α, PPAR-γ, acetyl CoA carboxylase-1 (ACC-1), phosphorylated-ACC-1 (pACC-1), (Abcam, Cambridge, MA, USA), CPT-1, CPT2, AMPKα, phosphorylated AMPKα (pAMPKα), SREBP-1c, FAS, and sirt1, (Cell Signaling Technology, Danvers, MA, USA).

Lung tissue proteins were quantified and separated on 10% SDS polyacrylamide gels. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and incubated with primary antibodies overnight at 4°C. Then, membranes were washed and incubated with secondary antibodies. Finally, PVDF membranes were treated with Luminol/Enhancer Solution (Millipore) to detect antibody signals with the BioSpectrum 600 system (UVP, Upland, CA, USA). Primary antibodies included anti-HO-1, anti-Nrf2, and anti-Lamin B1 (Santa Cruz, CA, USA); β-actin expression was evaluated as a loading control (Sigma).