Ab119716

A Western blot of mice hearts and CFs was performed as previously described (60 (link)). The antibodies used in this study are as follows: Smad2/3 (CST, 8685S, 1:1,000), phospho-SMAD2 (CST, 18338, 1:1,000), phospho-SMAD3 (CST, 9520, 1:1,000), and β-actin (Abcam, ab119716, 1:1,000). ImageJ software was used to quantify the intensities of the protein bands.

Total protein was extracted from the L4-L6 spinal cord segments as previously described66 (link). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-GFAP (1:500) (ab53554, Abcam), rabbit anti-Substance P (1:500) (orb215527, Biorbyt), rabbit anti-CGRP (1:500) (ab47027, Abcam), rabbit anti-PDGF BB (1:300) (ab16829, Abcam), rabbit anti-pPDGFRα (1:1000) (#2992, Cell Signal), rabbit anti-pPDGFRβ (1:1000) (#2227,Cell signal), rabbit anti-ERK(1:500) (ab184699, Abcam), rabbit anti-pERK (1:1000) (#4370, Cell Signal), rabbit anti-AKT (1:500) (9272s, Cell Signal) and rabbit anti-pAKT (1:1000) (4056s, Cell Signal). The membranes were subsequently incubated with the appropriate secondary antibodies (1:5000; Abcam) for 1 h at room temperature. The protein bands were detected using an enhanced chemiluminescence detection system. Then, the membranes were stripped with stripping buffer (P0025, beyotime) and reblotted with antibodies against the loading controls [mouse anti-β-actin (1:5000) (ab119716, Abcam) and mouse anti-GAPDH (1:5000) (ab9484)]. The intensity of the immunoreactive bands was quantified using Image J software. The ratio of target protein to β-actin (loading control) levels was statistically analyzed. Western blot experiments were repeated 3 times.

Protein was extracted using lysis buffer (Thermo Fisher), and the concentration was evaluated using a bicinchoninic acid kit (Pierce, USA). Equal amounts of protein were taken from each sample, and transferring was performed with 10% SDS-PAGE (Bio-Rad, USA) using the semi-transfer method after the protein was mixed with loading buffer. The membrane was probed with primary antibodies to Col II (1:10000, ab34712, Abcam), aggrecan (1:100, ab36861, Abcam), Bax (1:1000, #14796, Cell Signaling Technology, USA), Bcl-2 (1:2000, #33-6100, Thermo Fisher), and β-actin (1:5000, ab119716, Abcam) overnight at 4°C. After tris-buffered saline-Tween (TBST) washing, the membrane was incubated with secondary antibodies goat anti-rabbit IgG H&L (HRP) (1:50000, ab205718, Abcam) and goat anti-mouse IgG H&L (HRP) (1:10000, ab205719, Abcam) at room temperature for 2 h, followed by one TBST washing. Proteins were detected by chemiluminescence (Millipore, USA).

The HEI-OC-1 cell line and cochlear tissue were lysed with ice-cold RIPA lysis buffer (PP109, Protein Biotechnology) plus Phosphatase Inhibitor Cocktails (04693132001, Roche) for 30 min at 4°C. Protein concentrations were determined using a BCA Protein Quantification Kit (PP202, Protein Biotechnology) according to the manufacturer’s instructions. Equal amounts of protein were loaded onto a 12% Tris-glycine SDS-PAGE gel, separated at 120 volts for 1.5 to 2 h, and then transferred to a nitrocellulose membrane and blocked with 5% milk in PBST [1 × PBS with 0.1% Triton X-100 (Solarbio, 1109F0521)] buffer. Cleaved caspase 3 was evaluated using anti-cleaved caspase 3 rabbit monoclonal antibody (1:1,000 dilution, 9664S, Cell Signaling Technology), and β-actin was measured using a mouse monoclonal antibody (1:5,000 dilution, ab119716, Abcam, Cambridge, United Kingdom). Peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G (ab6789 and ab6721, Abcam) was used as the secondary antibody. The proteins were detected using a SuperSignal West Dura chemiluminescent substrate kit (34075, Thermo Scientific) according to the manufacturer’s instructions. Semi-quantification of the western blot results was done by measuring the intensities of the bands using ImageJ.

For immunoblots, we homogenized and sonicated tissues and cells in cell lysis buffer (6 (link)) with protease inhibitors (Sigma-Aldrich). Lysates were run under denaturing conditions on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Blots were probed with anti-SNAP23 (1:5000; Abcam: ab3340), anti-SNAP25 (1:50,000; Synaptic Systems: 111002), anti-SNAP29 (1:10,000; Abcam: ab138500), anti-SNAP47 (1:20,000; Synaptic Systems: 111403), anti-β-actin (1:20,000; Abcam: ab119716), and anti-GAPDH (1:30,000; Abcam: ab9483) antibodies.

Total protein from tumor tissues and cultured cells were lysed in RIPA buffer with protease inhibitor (Beyotime, Shanghai, China). The protein was quantified using a BCA assay kit (Beyotime, Shanghai, China). A total of 20 μg of total protein were separated by 10 % SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and then reacted with primary antibodies against IL-37 (24 kDa, ab101376, 1:500), and β-actin (42 kDa, ab119716, 1:1000) (all from abcam, Cambridge, UK). After being extensively washed with PBS containing 0.1 % Triton X-100, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody for 30 min at room temperature. The bands were visualized using 1-step TM NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL) and detected by an Alpha Imager (Alpha Innotech, San Leandro, CA).