Small molecule, indirubin-3-oxime, for prevention and treatment of bone disease
a small molecule, bone disease technology, applied in the direction of skeletal disorder, food preparation, medical preparation, etc., can solve the problems of inconvenient use, no drug has a direct target for height growth, and growth hormone injection is not suitable for a child with normal hormone secretion, etc., to achieve low toxicity, low development cost, and clear
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example 1
Cell Culture and Reagents
[0026]Rat chondrosarcoma (RCS) cells were cultured in a Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, N.Y.), supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin.
example 2
Western Blot Analysis
[0027]For Western blot analysis, cells were treated with indirubin-3′-oxime for 24 hours, cultured and reached to 70% confluency and then were harvested. After the harvested samples underwent cytolysis, the proteins were quantified, which was followed by electrophoresis using 10% or 12% SDA-PAGE gel. Afterwards, immunoblot was conducted using antibodies.
example 3
[0028]2.5×104 cells were adhered to a cover glass placed in a 12-well plate. Once securely attached, cells were treated with indirubin-3′-oxime for 24 hours, and then were rinsed with a phosphoric acid buffer solution. After adding 4% paraformaldehyde, cells were fixed at room temperature for 20 min. Then, proteins expressed in the cells were observed through cell membrane permeation, blocking, and immunofluorescent staining processes.
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