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Published in Chad A. Mirkin, Spherical Nucleic Acids, 2020
Hui Li, Bohan Zhang, Xueguang Lu, Xuyu Tan, Fei Jia, Yue Xiao, Zehong Cheng, Yang Li, Dagoberto O. Silva, Henri S. Schrekker, Ke Zhang, Chad A. Mirkin
SKOV3 cells were plated in a 24-well plate at a density of 1.0 × 105 cells per well and cultured for 24 h. Then medium was replaced with serum-free DMEM immediately before treatment with free DNA (200 nM), POSS SNA (500 nM), C60 SNA (500 nM, 1000 nM), and Lipofectamine-complexed DNA (200 nM, following manufacturer suggested protocol). After 15 h, the medium was replaced with fresh, full growth medium, and cells were cultured for another 48 h. Whole cell lysates were prepared in 75 μL of radioimmunoprecipitation assay (RIPA) Cell Lysis Buffer (Sangon Biotech). Protein concentrations were determined using a bicinchoninic acid assay (BCA) Protein Assay Kit (Sangon Biotech). Equal amounts (25 μg) of protein samples were fractionated by 4 to 20% precast gradient gels (Beyotime), transferred to polyvinylidene difluoride (PVDF) membrane, and blocked with 5% nonfat milk in tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature (Bio-Rad). Proteins were analyzed by Western blotting with rabbit primary antibodies against HER2 (1000:1) (Cell Signaling), rabbit primary antibodies against GAPDH (1000:1) (Sangon Biotech), and HRP-conjugated goat anti-rabbit IgG secondary antibodies (2000:1) (Sangon Biotech) using an ECL Western blotting substrate (Beyotime).
Chitosan oligosaccharide promotes osteoclast formation by stimulating the activation of MAPK and AKT signaling pathways
Published in Journal of Biomaterials Science, Polymer Edition, 2018
Bing-Li Bai, Zhong-Jie Xie, She-Ji Weng, Zong-Yi Wu, Hang Li, Zhou-Shan Tao, Viraj Boodhun, De-Yi Yan, Zi-Jian Shen, Jia-Hao Tang, Lei Yang
To examine the signaling pathways affected by COS, BMMs were seeded in 6-well plates at a density of 5 × 105 cells/well as described previously [18]. After reaching confluence, the cells were pretreated with different doses of COS (0,5, 50 or 500 ng/ml) for 4 h, and then were stimulated with 50 ng/ml RANKL for different time periods. Afterward, the cells were washed twice with PBS and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich). Lysates were centrifuged at 12,000 × g for 15 min and the supernatants were collected. Protein concentrations were determined using the bicinchoninic acid assay. Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, U.S.A.). The membranes were blocked in 5% non-fat dry milk in TBST [50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20] at room temperature for 1 h and incubated with the primary antibodies overnight at 4 °C. Protein bands were visualized using LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan), and the obtained images were analyzed with the ImageJ software.