Ab246916

Cellular proteins were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined by the BCA method. A total of 40 μg of protein for each group was separated on 12% SDS-PAGE gels and transferred to 0.45-μm PVDF membranes (EMD Millipore). Membranes were then blocked with 5% BSA and incubated with primary antibodies at 4°C overnight against HPS70 (4876, CST), CD9 (ab92726, CST), CD63 (ab193349, Abcam), RBP4 (ab233138, Abcam), RPSA (ab133645, Abcam), RPS3(9538, CST), RPS20 (ab133776, Abcam), RPS14 (ab246916, Abcam), RPL4 (ab234829, Abcam), RPL13 (ab134961, Abcam), HSPD1 (ab46798, Abcam), HSPA8 (8444, CST), P-gp (13342, CST), p-cofilin-1 (3313, CST), cofilin-1 (5175, CST), PP1 (sc-7482, Santa Cruz), PP2A (9780, CST). Anti-β-actin (ab179467, Abcam) and anti-GAPDH (2118, CST) were used as the internal control. Anti-COX IV (11967, CST) was used as loading control for mitochondrial proteins. The membranes were then incubated at 37°C for 1 h with an HRP-conjugated secondary antibody (ab97051, Abcam). Bands were visualized by chemiluminescence according to the manufacturer’s protocols.

Cochleae were dissected and fixed in 4% paraformaldehyde (PFA) at room temperature for 2 h. After fixation, the samples were decalcified in 0.5 mM ethylene diamine tetraacetic acid (EDTA, Biosharp, #BL518A) and then cut into several pieces from the apical, middle and basal turns. Spheres and organoids were fixed with 4% PFA at room temperature for 1 h. Samples were blocked with 0.5% (vol/vol) Triton X‐100/10% (vol/vol) donkey serum in 1× phosphate buffered saline (PBS), at room temperature for 1–2 h. After blocking, the samples were stained with antibodies against Myosin 7A (Proteus Biosciences, #25‐6790, 1:1000 dilution), Sox2 (Santa Cruz Biotechnology, #sc‐17,320, 1:200 dilution) and RPS14 (Abcam, #ab246916, 1:100 dilution) together with corresponding secondary antibodies and 4′6‐diamidino‐2‐phenylindole (DAPI). EdU detection was performed following the instruction of the Click‐iT® Plus EdU Imaging Kit (ThermoFisher, #C10640). Finally, the samples were immersed in DAKO Fluorescence Mounting Medium (DAKO, #S3023) and observed by confocal microscopy (Zeiss LSM 900).