Precast polyacrylamide gel

Frozen pellets were thawed on ice and then lysed by resuspending in 10 mL of lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole; pH 7.4) containing 1 mg/mL lysozyme (Sigma) and 25 U/mL Benzonase^® Nuclease (Merck Millipore, Burlington, MA, USA) and incubated on ice for 30 min. Supernatant, containing soluble proteins, was separated from non-lysed bacteria and cell debris by high speed centrifugation (14,000× g, 30 min) at 4 °C. Supernatant was loaded onto a gravity-flow Fast Start Column (QIAGEN, Hilden, Germany) packed with 0.5 mL of Ni-NTA (nickel–nitrilotriacetic acid) resin and pre-equilibrated with 10 column volumes of buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole; pH 7.4). Each column was then washed twice with 4 mL of wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 50 mM imidazole; pH 7.4), followed by elution of the bound nanobody–His(6) proteins with elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 750 mM imidazole; pH 7.4) in two 1 mL fractions. Samples from individual washing and elution steps were analyzed on SDS-PAGE: 5 µL of sample was mixed with 10 µL ultra-pure water and 5 µL Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) and 2-mercaptoethanol was added to a final concentration of 355 mM. The samples were boiled for 10 min and then loaded onto 16.5% precast polyacrylamide gels (Bio-Rad) and run using a Mini-PROTEAN Tetra Cell electrophoresis system (Bio-Rad). Protein bands were visualized on gels by incubation overnight in InstantBlue Protein Stain (Expedeon, Cambridge, UK). Eluted fractions had their buffers exchanged to HEPES buffer (20 mM HEPES, 115 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mM K₂HPO₄, pH 7.4) and were pooled and concentrated using 3 kDa centrifugal filter units (Amicon Ultra 0.5 mL, Merck Millipore). Subsequently, concentrated nanobody–His(6) fractions were loaded at a flow rate of 0.8 mL/min onto a Gel-filtration column (Superdex 75 10/300 GL, GE Healthcare Life Sciences, Chicago, IL, USA) pre-equilibrated with 48 mL of 20 mM HEPES buffer, pH 7.4. Fractions of 0.5 mL, corresponding to observed peaks at 280 nm and 214 nm, were collected and analyzed for size and purity on SDS-PAGE (see Supplementary Materials, Figure S1). Pure fractions corresponding to the size of monomeric nanobody–His(6) were pooled and concentrated using 3 kDa centrifugal filter units (Amicon Ultra-0.5 mL, Merck Millipore), and final protein concentration was determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific). Protein fractions were kept at −80 °C until further use.

For total protein extraction, sensory inner ear neuron cultures from hiPSCs-derived ONPs from day 0 to day 21 were lysed using cell lysis buffer 10 × (Cell Signaling Technology, USA) containing protease inhibitor cocktail (Sigma Aldrich, USA). Cell lysates were separated by molecular weight using precast polyacrylamide gels (Bio-Rad, USA) and transferred to nitrocellulose membranes (Bio-Rad, USA). Proteins were detected using the ChemiDoc XRS + System (Bio-Rad, USA). To detect ANK2, the ankyrin B polyclonal antibody (PA5-82,326, Invitrogen) was used. GAPDH (SAB3500247, Sigma Aldrich) loading control for total protein extractions was used. Western blotting was carried out using standard procedures. Image processing and quantification of band intensity were executed using Image Lab software (Bio-Rad, USA).

Total protein was isolated from cell pellets lysed in RIPA buffer (1x PBS, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) supplemented with EDTA 1 mM and a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was determined with the Pierce BCA™ protein assay kit (Thermo Fisher Scientific). For Western blotting, 10–30 µg of total protein extracts were subjected to electrophoresis on 4–15% polyacrylamide precast gels (Bio-Rad, Hercules, CA, USA) and blotted onto 0.2 µm PVDF membranes (Trans-Blot Turbo Mini, Bio-Rad). The membranes were blocked with 5% skim milk powder in 1x Tween-Tris-buffered saline (TTBS) and subsequently incubated with the corresponding primary antibodies diluted in the same buffer overnight at 4 °C. The next day, the membranes were washed in 1x TTBS, incubated with the secondary HRP-conjugated antibody, and washed again. The membranes were finally incubated with enhanced chemiluminescence (ECL) reagents (Merck Millipore, Darmstadt, Germany) and photographed on an Amersham ImageQuant™ 800 (Cytiva). The membranes were incubated sequentially with different antibodies after stripping for 30 min at 65 °C with stripping buffer (Tris-HCl, pH 6.8; SDS, 10%; β-mercaptoethanol, 0.7%). The primary antibodies used were as follows: anti-FLI1, 1:500 (#133485, Abcam); anti-DAX1 (NR0B1), 1:1 000 (2F4 clone) (a generous gift of Dr. E. Lalli; [22 (link)]); anti-CD44, 1:1 000 (#ab51037, Abcam); and anti-β tubulin, HRP-conjugated, 1:10 000 (#ab185057, Abcam). Anti-mouse m-IgG-HRP (#sc-516102) and anti-rabbit IgG-HRP (#sc-2357) secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).