Prior to ELISA and western blot analysis, cells in both the 2D and 3D cell culture formats were lysed in ice-cold RIPA buffer (20 min, frequent agitation, 4 °C, 2 × 106 cells per mL of lysis buffer). The resulting lysates were clarified by centrifugation at 10,000 ×g at 4 °C for 10 min. For ELISA, ERα and HIF-1α concentrations were determined with commercial kits (R&D Systems, DYC-5715 and DYC1935); β-actin concentration was determined with a sandwich assay consisting of a β-actin capture antibody (R&D Systems, AF4000) and an HRP-linked detection antibody (SantaCruz Biotechnology, sc-47778).
For western blot analyses, clarified lysates were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with antibodies for ERα (1:500), HIF-1α (1:200), and β-actin (1: 500), detected with HRP-conjugated secondary antibodies (1: 1000 m-IgGº BP-HRP), and visualized with the Pierce ECL Plus Western blotting substrate. Antibodies for ERα (sc-8002), β-actin (sc-47778), and mIgGº BP-HRP (sc-516102) were purchased from Santa Cruz Biotechnology; the HIF-1α antibody (610958) was purchased from BD Transduction Laboratories.
For western blot analyses, clarified lysates were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with antibodies for ERα (1:500), HIF-1α (1:200), and β-actin (1: 500), detected with HRP-conjugated secondary antibodies (1: 1000 m-IgGº BP-HRP), and visualized with the Pierce ECL Plus Western blotting substrate. Antibodies for ERα (sc-8002), β-actin (sc-47778), and mIgGº BP-HRP (sc-516102) were purchased from Santa Cruz Biotechnology; the HIF-1α antibody (610958) was purchased from BD Transduction Laboratories.