Alkaline phosphatase alp activity kit

Mouse IL-17A was from Peprotech Inc. (Rocky Hill, NJ, USA). Dexamethasone, L-ascorbic acid, β-glycerophosphate, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). All cell culture media and supplements were from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reagents for the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were obtained from Takara Bio, Inc. (Otsu, Japan). MTT was purchased from Amresco, LLC (Solon, OH, USA). Rabbit anti-PI3K (cat. no. 4292), anti-phosphorylated (p)-PI3K (cat. no. 4228) and anti-GAPDH (cat. no. 2118) monoclonal antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Goat anti-rabbit immunoglobulin G secondary antibodies (cat. no. BA1054) were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Enhanced chemiluminescence (ECL) detection reagent was purchased from Thermo Fisher Scientific, Inc. Alkaline phosphatase (ALP) activity kit was provided by Beyotime Institute of Biotechnology (Haimen, China).

Icaritin (>98% purity) was purchased from Shanghai U-sea Biotech Co. Ltd. modified Eagle's medium of alpha (α-MEM) and fetal bovine serum (FBS) were both obtained from Hyclone (Hyclone; GE Healthcare Life Sciences); The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. Dimethyl sulfoxide (DMSO), trypsin and TRIzol^® reagent were purchased from Gibco; Thermo Fisher Scientific, Inc.; the alkaline phosphatase (ALP) activity kit, enhanced chemiluminescent detection reagent and micro-BCA assay kit were obtained from Beyotime Institute of Biotechnology. DKK-1 and ICI182780 were purchased from R&D Systems, Inc. The RNA extraction kit was purchased from Takara Bio, Inc. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) primers were obtained from Invitrogen; Thermo Fisher Scientific, Inc. The antibodies used were obtained from Cell Signaling Technology, Inc., unless otherwise indicated. Other reagents used in the experiment were purchased from Sigma-Aldrich; Merck KGaA. Icaritin was dissolved in DMSO and the final concentration of DMSO was 0.05% (v/v). ICI 182780 was dissolved in DMSO and stored at 4°C. Cells were pretreated with ICI 182780 (1 µM) for 30 min at 37°C, followed by icaritin treatment.