Anti β actin antiserum

For cytokine array, flushed bone marrow cells were gently washed with serum-free RPMI 1640 (Gibco, Carlsbad, CA) media for 10 min at room temperature, and the media was recovered by centrifugation at 300 g for 5 min. 250 µg of total proteins measured by Bradford assay were applied to a mouse cytokine array (Raybiotech, Norcross, GA), as described previously (16). For western blotting, total tissue samples were solubilized in lysis buffer, and loaded, 20 µg per lane, on 12% SDS-PAGE. Proteins were blotted onto nitrocellulose membrane and probed using anti-TC1 antibody. Anti-β-actin antiserum was used for loading control (Sigma-Aldrich).

Total protein was extracted from uterine tissues according to the protocol, and the concentration of protein was confirmed with a Precision Red Assay (Cytoskeleton Inc.). Equal amounts of protein were stained with loading dye and separated with 12% SDS-PAGE. Protein was then transferred to polyvinylidene difluoride (PVDF) membrane and was incubated with 5% bovine serum albumin (BSA). The membrane was exposed to anti-VEGF antibody (1 : 500), anti-cytokeratin antibody (1 : 500), anti-vimentin antibody (1 : 500), anti-integrinα_ν antibody (1 : 300), anti-integrinβ₃ antibody (1 : 300), and anti-LIF antibody (1:300) overnight. The blots were washed with tris-buffer saline and incubated with secondary anti-rabbit IgG (1 : 3000; Cell Signaling Technology) for 1 hour at room temperature. The protein content was detected using SuperSignal West Pico (Pierce Biotechnology). Anti-β-actin antiserum (Sigma) was used as an internal standard between groups.