Goat anti ap 2δ

The expression levels of Prestin and AP-2δ protein in cells were detected by western blot. Protein samples were prepared in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, PMSF), dissociated on ice for 30 min and centrifuged at 12,000 rpm for 10 min at 4 °C. A total of 40–60 μg of supernatant was mixed with 5X loading buffer and electrophoresed on 10% SDS-PAGE then transferred to polyvinylidene fluoride (PVDF) membranes (Merk Millipore, USA). The membrane, blocked with 5% non-fat milk, was incubated with goat anti-Prestin (1:500; Santa Cruz, USA), goat anti-AP-2δ (1:1000; Santa Cruz, USA) and anti-GAPDH (11,000; CWBIO, China) at 4 °C overnight. Then, the corresponding secondary antibodies with HRP (15000) conjugates were added and incubated for 1 h at 37 °C. Finally, the signal was detected with BeyoECLPlus (Beyotime, China), analyzed by ImageJ software, and normalized for GAPDH staining.

HEI-OC1 cells were cross-linked with 1% formaldehyde for 10 min at 37 °C, the chromatin was prepared as described previously [28 (link)] and sheared into 200–600 bp fragments using a Bioruptor (Diagenod, Belgium). The samples of 100 μl each in a tube were diluted 10-fold in chromatin immunoprecipitation (ChIP) dilution buffer and incubated with 1 μg of goat anti-AP-2δ (1:1000; Santa Cruz, USA) or 1 μg of control nonimmune IgG at 4 °C overnight. Subsequently, the DNA-protein complexes were precipitated and purified as described by Heimann et al. [29 (link)]: 2 μl of IP DNA or input DNA were templated for SYBR PCR reactions using the primers flanking the identified Prestin transcriptional start site (TSS) -2000 − + 500 bp. The primer sequences (forward and reverse, respectively) were as follows: S-1441 ChIP-Prestin, 5-CTTGTGGGGTGAGGGTAGAA-3, 5-GGAGAAACTGGCTGTCTTGC-3; S-784 ChIP-Prestin, 5-TTGTGGATGCTGGCATTAGC-3, 5-TAAGCTTGAGCAGCAGGTG-3.