Ab82400

Each 100ng of recombinant human AGR2 and AGR3 protein (Biorbyt, Cambridge, UK) was diluted 1:2 in 2x NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 5% dithiothreitol and heat denatured (5min, 95°C). For separation protein was loaded on 4–12% gradient gels (NuPAGE; Invitrogen) and then transferred onto 0.2μm PVDF membranes (Whatman, Dassel, Germany) (1h, 100V) for immunodetection. Blots were blocked in TRIS-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% non-fat dry milk (Merck, Darmstadt, Germany) overnight at 4°C. Blocked blots were probed with mouse monoclonal anti-AGR3-antibody (ab82400, Abcam, Cambridge, UK), diluted 1:500 in blocking solution, for 1h at room temperature. After washing (TBS + 0.05% Tween-20), blots were incubated with rabbit anti-mouse (DAKO, Glostrup, Denmark) secondary peroxidase-conjugated antibody, diluted 1:4000 in blocking solution, for 1h at room temperature. After washing (TBS + 0.05% Tween-20), antibody detection was accomplished with Pierce ECL Western blotting Substrate (Thermo Scientific, Rockford, USA). Original uncropped blot is depicted in the supplements (S1 Fig.).

Immunohistochemical analysis was carried out according to the manufacturer’s instructions (DAKO 5001; DAKO, Glostrup, Denmark). Heat-induced epitope retrieval was performed in 10mM citrate buffer (pH 6.0) for 10 minutes using a pressure cooker. FFPE sections (3μm) were incubated for 45 minutes with mouse monoclonal anti-AGR3-antibody (1:1000; ab82400, Abcam, Cambridge, UK). AGR3 protein staining was quantified by a pathologist using an adapted immunoreactive scoring system (IRS) according to Remmele and Stegner [30 (link)].