Detection of Secreted and Cellular Proteins

For detection of secreted AGR3, conditioned media were collected after 48 h of cell culture in serum-free DMEM, centrifuged at 13,000 rpm for 10 min, followed by overnight precipitated with cold acetone (at 80% final concentration). For cellular proteins detection, cells were lysed in lysis buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na₃CO₄, 1% Nonidet P40) containing protease inhibitor cocktail and phosphatase inhibitor cocktail 2 (both Sigma-Aldrich). For detection of tyrosine-phosphorylated proteins, the aforementioned lysis buffer was supplemented with 100 µM sodium orthovanadate (Sigma). The primary antibodies used were as follows: Mouse monoclonal anti-tubulin (Sigma), goat monoclonal anti-actin (Santa Cruz Biotechnology), mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology), in-house mouse monoclonal anti-AGR3 (8 (link),25 (link)), Py-Plus™-HRP mouse anti-phosphotyrosine (Invitrogen), rabbit monoclonal anti-c-Src and anti-phospho-c-Src (both Cell Signaling Technology), mouse monoclonal anti-GAPDH (clone 6C5) (Millipore), mouse monoclonal anti-VCP (p97) (BD Biosciences) and rabbit monoclonal anti-EsRalpha antibody (Abcam).