Westernbright quantum

Western blots were performed as previously described (Sasaki et al., 2018 (link)). Sciatic nerves were quickly dissected and then placed in chilled PBS as the epineurium was removed. The nerves were then flash frozen and stored at −80°C. Sciatic nerves from postnatal day 7 mice were sonicated in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm EDTA, 10 mm NaF, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 1% NP-40, and 100 mm PMSF with protease inhibitors, Roche Diagnostics; and phosphatase inhibitors, Sigma-Aldrich). Lysates were centrifuged at 12,000 × g for 20 min at 4°C, supernatant was collected, and 10–20 μg of protein was boiled for 5 min in sample buffer with β-mercaptoethanol, loaded onto 10% SDS-PAGE gels, and then transferred onto nitrocellulose or PVDF membranes. Membranes were blocked in blocking solution (Super Block T20, Thermo Fisher Scientific) and then incubated with antibodies (Table 2) diluted in blocking solution. Membranes were washed, incubated in species-appropriate secondary antibodies (Cell Signaling Technology), and then developed using Western Bright Quantum (Advansta). Band intensity was measured using ImageJ (National Institutes of Health), normalized to α-tubulin or β-actin, and then displayed as fold change relative to the average of the WT samples.

Urinary mucosa, defined as urothelium and lamina propria, was surgically separated from the smooth muscle, homogenized using Lysing Matrix D in a FastPrep 24 instrument (MP Biomedicals, Solon, OH) following previously established protocols.^{14 (link)} Equal amounts of proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride membranes, which were then incubated overnight at 4°C with primary antibodies (Table 1) followed by secondary antibodies (donkey anti-rabbit horseradish peroxidase [1:5000] or sheep anti-mouse horseradish peroxidase [1:5000]; GE Amersham, Pittsburgh, PA) for 1 hour in 5% (w/v) milk TBS-T, incubated in WesternBright Quantum (Advansta, Menlo Park, CA) and then imaged on a ChemiDoc MP (Bio-Rad). As a loading control, total protein per sample was determined using Bio-Rad Stain Free SDS-PAGE gel technology (Bio-Rad). UV-activated protein fluorescence was imaged on a ChemiDoc MP (Bio-Rad). Assessment of protein carbonylation (as a marker for oxidative damage) was performed with an antibody that detects dinitrophenylhydrazine (DNPH)-derivatized carbonyl groups (OxyBlot Protein Oxidation Detection Kit, S7150; Millipore, Burlington, MA).