Ab134100

Human recombinant HGF (294-HGN-005) and GDNF (212-GD-010) were obtained from R&D Systems (Minneapolis, MN). Growth factor-reduced Matrigel (BD354230) was obtained from CORNING (Corning, NY), and atelocollagen (DME-02) was obtained from Koken (Tokyo, Japan). Antibodies used in this study were as follows: goat polyclonal anti-GDNF antibody (AF-212-NA) (R&D Systems); mouse anti-beta-actin antibody (3598R-100) (BioVision, Milpitas, CA); rabbit monoclonal anti-Ret antibody (E1N8X) (CST #14556) (Cell Signaling Technology, Danvers, MA); rabbit monoclonal anti-Ret antibody (ab134100) (Abcam, Cambridge, MA); anti-Met antibody (ab39075) (Abcam, Cambridge, MA); anti-phospho-Met antibody (CST #3077) (Cell Signaling Technology, Danvers, CO); anti-aquaporin 1 (AQP1) antibody (sc-20810) (Santa Cruz Biotechnology, Dallas, TX).

Total protein was extracted from HSCR ganglionic colon tissues with a radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors (cOmplete, ULTRA, 132Mini, EDTA-free, EASY pack; Roche, Basel, Switzerland). Protein samples were boiled at 95 °C for 5 min, cooled at room temperature for 5 min and then centrifuged. Protein concentrations were determined using the BCA (bicinchoninic acid) method. Equal amounts of protein (50 μg) were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Roche, Basel, Switzerland). Membranes were blocked using 5% skimmed milk and incubated with the appropriate diluted antibodies (anti-NRG1, 1:1000, #ab27303; anti-RET, 1:1000, #ab134100; Abcam, Cambridge, UK). Membranes were subsequently incubated with a 1:4,000 dilution of a horseradish peroxidase-conjugated secondary antibodies (Invitrogen, Logan, UT, USA) for 1 h, followed by 3 washes with TBST for 15 min. Membranes were then processed using enhanced chemiluminescence (ECL) (Bio-Rad, Hercules, CA, USA) and were exposed to film (Canon, Tokyo, Japan). The experiments were repeated 3 times. The protein expression levels of NRG1 and RET were normalized to those of GAPDH. All the experimental procedures were conducted according to the manufacturer’s recommended instructions.

Immunohistochemistry analysis (IHC) was performed on thin sections (3 μm) of previously formalin-fixed and paraffin-embedded tissues. The antibody used was monoclonal rabbit anti-human RET (ab134100; Abcam Inc., Cambridge, MA, USA), Sections representing MTC were submitted to routine immunohistochemical technique, which comprises deparaffination and rehydration, antigenic recovery, inactivation of endogenous peroxidase, and blockage of unspecific reactions. Primary antibodies were incubated overnight at a temperature of 4°C, at dilutions of 1:400 followed by application of streptavidin horseradish peroxidase conjugate (LSAB; DakoCytomation, Via Real Carpinteria, CA, USA), and diaminobenzidine tetrahydrochloride (Kit DAB; DakoCytomation). Absence of the primary antibody was used as a negative control.