Tumor-containing tibiae and subcutaneous tumors were snap-frozen in liquid nitrogen. Frozen tibiae were homogenized in RIPA buffer (50 (link)) using a FastPrep-24 tissue homogenizer (MP Biomedicals). Prostate cancer cell lines were lysed using RIPA buffer as described previously (50 (link)). Total protein (10–20 μg) was separated by SDS-PAGE and transferred to PVDF membranes (Fisher). Membranes were blocked with 5% BSA-TBST and incubated with antibodies diluted in 5% BSA-TBST as previously described (50 (link)). Primary antibodies were detected using HRP-conjugated secondary antibodies (Sigma) by chemiluminescence using Quantity One imaging software on a Bio-Rad Gel Docking system. Antibodies listed in Supplementary Table S1. Equal amounts of protein from subcutaneous or tibiae tumors pooled from two mice were incubated with a 174-protein spotted human cytokine antibody array C2000 (Ray Biotech Inc) according to the manufacturer’s protocol.
An MMP-3 ELISA (R&D) that recognizes both the proenzyme and active form was performed on 24 h serum-starved PC3-NICD3 cells treated with or without Dox. Media were collected and concentrated (Ultracel-10k; Millipore) and equal volumes were used to quantify total MMP-3.
An MMP-3 ELISA (R&D) that recognizes both the proenzyme and active form was performed on 24 h serum-starved PC3-NICD3 cells treated with or without Dox. Media were collected and concentrated (Ultracel-10k; Millipore) and equal volumes were used to quantify total MMP-3.