Phospho foxo1

Whole-cell lysates of mouse LV and NRVMs were prepared in T-PER (Thermo) containing protease and phosphatase inhibitors (Roche). Nuclear fractions of heart 4 days following sham- or TAC procedures were prepared using NE-PER nuclear cytoplasmic extraction reagents according to the manufacturer’s instructions (Thermo). Equal amounts of protein (3–10 μg) were separated on a 4–20% SDS-PAGE gel (Bio-Rad), transferred to a nitrocellulose membrane, and immunoblotted to detect and quantify specific protein bands using an Odyssey scanner (LI-COR version 3)^{74 (link)}. Proteins were detected with a 1000-fold dilution of the following primary antibodies: FoxO1 (#2880, Cell Signaling and ab39670; Abcam); Phospho-FoxO1 (#9464, Cell Signaling), ERK (#4695, Cell Signaling); Phospho-ERK (#4370, Cell Signaling); 10,000-fold dilution of GAPDH (10R-G109a, Fitzgerald) and tubulin (ab6046; Abcam) and 500-dilution of Dio2 antibody (ab77481, Abcam) antibodies, respectively.

Chromatographically pure Sch was purchased from Feng-Shan-Jian Medicine Research Co. Ltd. (Kunming, Yunnan, China) (Structure shown in Figure 1), and dissolved in 0.5% dimethyl sulfoxide (DMSO) at a concentration of 0.5 mg/mL before experiment. LPS (Escherichia coli serotype O55:B5), Evans blue (EB) dye, and rhodamine 6G were purchased from Sigma Chemical (St. Louis, MO, United States). Primary antibodies against I-κBα, phospho-I-κBα, NF-κB p65, Erk1/2, phospho-Erk1/2, p38 MAPK, phospho-p38 MAPK, phospho-FoxO1, Akt, phospho-Akt, Keratin, caspase-3, cyclin D1, cyclin B1, p27^kip1, phospho-retinoblastoma protein (Rb), GAPDH, and histone H3 were obtained from Cell Signaling Technology (Beverly, MA, United States). Antibodies against Claudin-5, Occludin and ZO-1 were from Invitrogen (Rochford, IL, United States), antibodies against FoxO1, p21^cip1, myeloperoxidase (MPO) and von Willebrand factor (vWF) from Abcam (Cambridge, United Kingdom). Antibody against VE-Cadherin from Santa Cruz Biotechnology (Santa Cruz, CA, United States), antibody against TLR4 from Novus Biologicals (Littleton, CO, United States) and antibody against prosurfactant protein C (proSP-C) from EMD Millipore Corporation (Temecula, CA, United States).

Total protein was prepared from cultured GCs or ovarian tissue as previously described.^{19 (link)} Nuclear and cytoplasmic proteins were extracted using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo) according to the manufacturer’s instructions. Immunoblotting was performed with primary antibodies against caspase-3 (1 : 1000; Cell Signaling Technology, #9662s, Danvers, MA, USA), cleaved caspase-3 (1 : 500; Cell Signaling Technology, #9661s), FoxO1 (1 : 1000; Cell Signaling Technology, #2880), Ac-FKHR (1 : 500; Ac-FoxO1, Santa Cruz Biotechnology, sc-49437), phospho-FoxO1 (Ser 256, 1 : 1000; Cell Signaling Technology), phospho-FoxO1 (Ser 319, 1 : 1000; Bioworld Technology, BS4712, MN, USA), FasL (1 : 500; Bioworld Technology, BS1122), Bcl-2 (1 : 1000; Proteintech, 12789-1-AP, Wuhan, China), Bax (1 : 500; Bioworld Technology, BS6420), and SIRT1 (1 : 500; Santa Cruz Biotechnology, sc-15404). β-actin (1 : 10000; Abcam, Cambridge, CA, USA), HSP60 (1 : 1000; Bioworld Technology), and Lamin B (1 : 1000; Santa Cruz Biotechnology, sc-6216) served as internal controls for detecting the expression levels of total protein lysates, cytoplasmic protein lysates, and nuclear protein lysates, respectively.

Penicillin-streptomycin was obtained from Lonza (Walkersville, MD, USA). The EZ-Cytox Cell Viability Kit was purchased from Daeil Lab (Seoul, Korea). LA, N-acetyl-l-cysteine (NAC), 2′,7′-dichlorofluorescein diacetate (DCF-DA), ortho-phthaldialdehyde (OPA), chloroform, and trypsin-EDTA solutions were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from Gibco Life Technologies Inc. (Rockville, MD, USA). Antibody against HSP70 was purchased from Enzo Life Sciences (Aargau, Switzerland). Antibodies against Bcl-2, Bax, phospho-FoxO1, β-actin, anti-rabbit IgG, and anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SOD1 and MuRF1 were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Vectashield mounting medium containing DAPI was purchased from Vector Laboratories (Burlingame, CA, USA). HPLC-grade methanol and HPTLC silica gel plates were purchased from Merck (Darmstadt, Germany). Ceramide, sphingomyelin, dihydrosphingomyelin, and sphingolipid ceramide N-deacylase (SCDase) were purchased from Avanti Polar Lipid Inc. (Alabaster, AL, USA). All other chemicals were commercially available.