Ta811000

Four wildtype mice and 3 Nav1.9 knockout mice were included in WB assay. Protein lysate from bilateral cochlea samples for each mouse was prepared in RIPA lysis buffer with the adding of complete protease inhibitor by Tissue Grinding Pestles, followed by keeping in an ice bath for 10 min. After centrifugation at 12,000 rpm for 10 min at 4 ℃, the supernatant mixed with loading buffer was denatured and loaded on a SDS-PAGE (12%) gel. Proteins were transferred to PVDF membrane for 90 min in an ice bath. After blocking, the membrane was incubated in primary antibodies of anti-Nav1.9 and anti-β actin with gentle agitation at 4 ℃ overnight, followed by HRP-conjugated secondary antibody incubation. Finally, the signal of HRP was detected using GE Healthcare’s ECL detection reagent. Antibodies in this study were as follows: polyclonal rabbit anti-SCN11A polyclonal antibody (AT322395, 1:1000, OriGene), beta Actin mouse monoclonal antibody (TA811000, 1:1000, OriGene), anti-rabbit HRP-linked IgG (7074, 1:4000, Cell Signaling Technology), goat anti-mouse HRP-linked IgG (H+L) (LK2003, 1:4000, sungene biotech).

For Western blot experiments we used anti-BCL6 antibody (Santa Cruz Biotechnology, sc-7388 & sc-550543, 1:250), anti-RAG1 (Abcam, ab172637, 1:2500), anti-RAG2 (Proteintech, 11825-1-AP, 1/1000) and β-Actin as a loading control (ORIGENE, TA811000, 1:500).

Antibodies against FLAG (F1804, Sigma Aldrich), IPMK (custom rabbit polyclonal antibody, raised against a mouse IPMK peptide corresponding to amino acids 295–311 [SKAYSTHTKLYAKKHQS; Covance]; Kim et al., 2011 (link)), SMARCB1 (A301-087, Bethyl), BRG1 (ab110641, Abcam), BAF155 (11956, Cell Signaling Technology), BAF170 (12760, Cell Signaling Technology), BAF250A (12354, Cell Signaling Technology), BRM (11966, Cell Signaling Technology), PBAF/PBRM (A301-591A, Bethyl), a-TUBULIN (T5169, Sigma Aldrich), GST (2622, Cell Signaling Technology), LaminB1 (sc-365214, Santa Cruz Biotech), histone H3 (05–499, Sigma Aldrich), GAPDH (sc-32233, Santa Cruz Biotech), anti-DPF2 (ab128149, Abcam), anti-SMARCE1 (ab137081, Abcam), anti-SS18L1 (ab227535, Abcam), anti-ACTL6A (sc-137062, Santa Cruz Biotech), anti-SMARCD1 (sc-135843, Santa Cruz Biotech), anti-BCL7A (HPA019762, Atlas Antibodies), and anti-ACTB (TA811000, Origene) were used for immunoblotting. Antibodies against IPMK (homemade), SMARCB1 (A301-087, Bethyl), and rabbit IgG isotype control (02–6102, Invitrogen) and anti-FLAG M2 affinity gel (A2220, Sigma Aldrich) were used for immunoprecipitation, and GST (2622, Cell Signaling Technology) was used for pull-down assays. Antibodies against FLAG (F7425, Sigma Aldrich), BRG1 (ab110641, Abcam), and IgG (homemade) were used for CUT&RUN assays.