Anti β catenin sc 7199

Seven micrometer thick sections were cut parallel to the long axis of the tibia using a CM1850 cryostat at −30°C (Leica) and mounted on Superfrost Plus slides (Menzel-Glaser). Sections were hydrated with decreasing concentration of ethanol, to remove OCT, fixed in neutral buffered formalin solution and washed with 0.5 M glycine in PBS. Sections were digested with 1 mg/ml hyaluronidase from sheep testes (Sigma–Aldrich) in 0.15 M sodium chloride, 0.1 M sodium acetate, pH 5.5, for 45 min at 37°C. 5% BSA, 0.05% Tween-20 (v/v) in PBS was used for blocking, and 0.05% Tween-20 (v/v) in PBS for washing steps. The following primary antibodies diluted 1:100 in 1% BSA, 0.05% Tween-20 in PBS were used: anti-β-catenin (sc-7199, Santa Cruz Biotechnology, Inc.) and anti-phospho-β-catenin (Ser33/37/Thr41, 9561, Cell Signaling Technology). Secondary antibody was Alexa Fluor® 647 goat anti-rabbit (Life Technologies). The nuclei were counterstained by 0.5 μg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma–Aldrich). Slides were mounted using Mowiol (Sigma–Aldrich). The sections were examined by TCS SP2-Leica confocal microscope (Leica). Co-localization was evaluated on single planes.

HDM, 100000 SQ-U (U)/ml in Alutard, was purchased from ALK-Abello A/S (Guangzhou, China). 1G6-D7 (noncommercial antibody), the anti-secreted Hsp90α mAb, was kindly granted by the University of Southern California Keck School of Medicine in USA. The Rho kinase inhibitors, Y-27632 2HCl (S1049) and GSK429286A (S1474), were obtained from Selleck Chemicals (Shanghai, China). Human recombinant (hr) Hsp90α (SPR-101C) and hrHsp90β (SPR-102C) were purchased from Stressmarq Bioscience (Victori, Canada). Anti-β-actin(8457), anti-MLC(3672), anti-phospho(p)-MLC(3674) antibodies and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit or anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Beverly, USA). Anti-Hsp90αantibody (CA1023) was obtained from Calbiochem (Billerica, USA) and anti-Hsp90β antibody (SMC 107) was from Stressmarq Biosciences (Victoria, Canada). Anti-E-cadherin (sc-7870) and anti-β-catenin (sc-7199) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, USA). Alexa 488-labeled goat anti-rabbit secondary antibody was from Life Technologies (Grand Island, USA).