Easypack

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.

Raw264.7 cells were cultured in the presence of sRANKL (100 ng/mL) with or without each PMF for 15 min. Raw264.7 cells were lysed in a lysis buffer containing PhosSTOP (Roche) and complete protease inhibitor cocktail EASYPack (Roche). The whole cell lysates were centrifuged at 12,000× g for 10 min and the supernatant was collected. The protein concentration of the supernatant was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc.). The 10 μg of protein in each sample was applied to SDS-PAGE with 10% polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Merck KGaA.). Membranes were blocked with 5% dry milk in PBS-T (PBS with 0.05% Tween-20) and incubated with primary antibodies at 4 °C overnight. Membranes were incubated with the corresponding secondary antibody in 1% skim milk in PBS-T and developed with ECL prime Western blotting detection reagent (GE Healthcare Japan Corp.) by ChemiDoc XRS+ (Bio-Rad Laboratories Inc.). Primary antibodies against IκBα (35–41 kDa; Santa Cruz Biotechnology Inc.) and β-actin (43 kDa; Santa Cruz Biotechnology Inc.) were used.

After perfusion with saline fresh brains were frozen on dry ice and stored at − 80 °C. For protein extraction, the whole brain of postnatal day 6 (P6) animals or dorsal raphe tissue punches (adult animals) were homogenized with Passive Lysis Buffer (Promega) and proteinase inhibitors (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack, Roche) using an electrical homogenizer (POLYTRON PT 1200 E Manual Disperser, Kinematica). Tissue was further disrupted using a syringe and pipetting. The whole procedure was performed on ice. Samples were then shaken in a thermomixer at 1000 rpm and 4 °C in a cold room for 1 h, centrifuged, and supernatant collected. Samples were stored at − 80 °C until assaying. Luciferase assays (Firefly and Renilla) were done on the protein extracts according to manufacturer instructions (Dual-Luciferase Reporter Assay System, Promega). The signal was normalized to the amount of protein in the sample as determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer instructions.

pMBMECs from wild-type C57BL/6J mice, EpH4 and L-cells were lysed in RIPA buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA solution, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl and 1 mM PMSF], in the presence of protease inhibitor cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack (1 tablet/10 ml buffer; Roche Diagnostics, Mannheim, Germany). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific Pierce Protein Biology, Waltham, USA), according to the manufacturer's instructions. A total of 5–20 μg of each sample was loaded onto a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Protan, GE Healthcare, United Kingdom) using a Trans-Blot Turbo transfer system (BioRad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. Membranes were blocked with Rockland Buffer (Rockland, Limerick, PA, USA) for 1 h at room temperature and incubated overnight at 4°C with the primary antibodies (Table S1). On the following day, membranes were washed and incubated with secondary antibodies (Table S1), for 1 h at room temperature. Proteins were detected using an Odyssey near infrared imaging system and software (LI-COR Biotechnology, Lincoln, NE, USA). Band intensity was quantified using FIJI software (NIH, Bethesda, MD, USA) and normalized against β-actin intensity.

Cells were lysed in RIPA buffer supplemented with 1 mM Na₃VO₄, 1 mM cOmplete ULTRA Tablets, Mini, EDTA-free EASYPack (Roche), and 100 nM MG-132. Protein concentrations were measured using a DC Assay Kit (Bio–Rad), and 100 μg of protein was used for each sample. Samples were added to either mouse IgG magnetic beads (Cell Signaling) or to 6 μg of primary antibody and incubated at 4 °C overnight with gentle agitation. Samples containing antibody were added to cleared protein G beads (with active motifs) and incubated for 4 h at 4 °C with gentle agitation. All samples were then washed with TBS, and proteins were eluted from the beads by adding 2× nonreducing Laemmli buffer (250 mM Tris-HCl; 8% SDS; and 40% glycerol, pH 6.8) and incubation at 95 °C for 5 min. β-Mercaptoethanol was added to each sample, and immunoblotting was performed.