Western Blot Analysis of CADM1 in GIST

GIST-T1 cells, GIST-T1-CAD cells, a representative gastric GIST tissue, and a representative small intestinal GIST tissue were lysed in CelLytic M Cell Lysis Reagent (Sigma-Aldrich; Merck KGaA) containing 5 mM NAF, 1 mM Na₃VO₄, and proteinase inhibitor cocktail (Roche). As described previously (10 (link)), almost all gastric GISTs express a very low level of CADM1 protein and almost all small intestinal GISTs apparently express CADM1 protein. Western blot analysis was performed as previously reported (10 (link)). Briefly, anti-CADM1 chicken monoclonal antibody (clone. 3E1, MBL International), anti-KIT rabbit polyclonal antibody (A4502, Dako) or anti-β-actin mouse monoclonal antibody (ab8226, Abcam) were used for primary antibodies after electrophoresis and membrane transfer. Then, membranes were incubated either with horse radish peroxidase (HRP)-conjugated donkey anti-chicken IgY antibody (EMD Millipore, Sigma-Aldrich; Merck KGaA), HRP-conjugated goat anti-rabbit IgG antibody, or HRP-conjugated goat anti-mouse IgG antibody (Dako) after the electrophoresis and membrane transfer. Proteins of interest were then visualized by incubation with enhanced chemiluminescence (ECL) reagent (Promega).