Ab27303

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.

P3/P8 mouse cortices were lysed in Pierce RIPA buffer (Thermo Scientific) containing Complete protease inhibitors (Roche) and analyzed for protein content using Bradford reagent. A total of 20 μg of protein extract was separated on a NuPAGE 3%–7% Tris-Acetate gel (Life Technologies) and blotted onto nitrocellulose. Immunoblotting was performed using Abs against Nrg1-type1 (1:1,000 dilution; ab27303, Abcam) and β-actin (1:500; ab8226, Abcam). Immunoreactive bands were detected by enhanced chemoluminescence (GE Healthcare).