Protein marker

Lung tissues were lysed in RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mM NaCl, 10 mM Hepes, pH 7.3, 2 mM EDTA, and protease inhibitor mixture; Pierce), as we previously described 12 (link). After blocking, membranes were probed overnight at 4°C with the primary antibodies as follows: APJ (SC-33823; Santa Crutz, USA), Bax (14796; CST, USA), Caspase 3 (9662; CST, USA), Cleaved caspase 3 (9661; CST, USA), ɑ-SMA (14968; CST, USA), CD31 (3528; CST, USA), and GAPDH (2118; CST, USA). HRP-conjugated anti-rabbit or anti-mouse (CST) was used to detect the primary antibodies. After washing, the protein bands were visualized using an enhanced chemiluminescence (Bio-Rad). The relative expression of the proteins was quantified using densitometric scanning and analyzed by the Imaging J System and expressed as percent of controls. Molecular mass was determined relative to protein markers (BioRad).

Insulin, rosiglitazone, dexamethasone, HEPES, NaHCO₃, Oil Red O, sodium deoxycholate, and phenylmethylsulfonyl fluoride were obtained from Sigma-Aldrich (St Louis, MO, USA). SDS, Tween 20, and isopropanol were purchased from GENEray (Shanghai, China). Paraformaldehyde was obtained from TCI (Tokyo, Japan). All antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Western blotting loading buffer, Bio-Rad protein assay reagents, and protein markers were obtained from Bio-Rad (Hercules, CA, USA). Triglyceride (TG) and glucose detection kits were purchased from Asan Pharmaceutical (Seoul, Korea).

RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, and Dulbecco's Phosphate Buffered Saline (DPBS) were purchased from Gibco-BRL (Burlington, Ont, Canada). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33342, ribonuclease A (RNase A), and propidium iodide (PI) were obtained from Sigma (St. Louis, MO, USA). DNA ladder size markers were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against Bcl-2, Bax, caspases 3 and 9, cleaved PARP, and β-actin were purchased from Cell Signaling Technology (Bedford, Massachusetts, USA). Protein marker was purchased from Bio-Rad (Richmond, CA, USA). Caspase 3 activity assay kit was purchased from Promega (San Luis Obispo, CA, USA). All other chemicals and reagents used herein were of analytical grade.

Referring to the previous description (32 (link)), protein extraction and immunoblotting were performed. The antibodies directed against the following proteins, including PI3K, p-PI3K, AKT, p-AKT, and β-actin were used, and their directions are shown in Table 1. HRP-conjugated secondary antibodies (1:5,000, GeneTex, Irvine, CA, USA) were also used. Other materials consisted of electrophoresis and membrane transfer device (Bio-Rad), RIPA lysate (Beyotime), protease inhibitor (cocktail) (Roche), ECL chemiluminescence substrate Kit (Biosharp, Hefei, China), defatted milk powder (Wondersun, Harbin, China), protein marker (Bio-Rad), BCA kit (Beyotime), and SDS-PAGE gel rapid preparation kit (Beyotime). Blotting was captured by Molecular Imager ChemiDocTM XSR+ Gel Imaging System (Bio-Rad) and analyzed using ImageJ software (NIH). In semiquantitative analysis of the target protein, each sample was normalized to β-actin.

Dry edible birds’ nest samples (Aerodramus fuciphagus) originated from Indonesia and were kindly donated by Xiamen Yanzhiwu Sinong Food Co., Ltd. (Xiamen, China). Edible birds’ nest samples with a higher content of protein (66.18 ± 0.76%) were milled by a high-speed universal crusher (SS-1022, Shengshun, Jinhua, China) and then passed through a 120-mesh sieve. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kit and protein marker were obtained from Bio-Rad (Hercules, CA, USA). 1-anilinonaphthalene-8-sulphonate (ANS) was obtained from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA).