Anti c fos

The detailed information of antibody is provided in Supplementary Table 1. Rabbit anti-A₁R antibody was purchased from Alomone labs (Jerusalem, Israel). Goat anti-A₁R, anti-c-Fos, anti-CRH, anti-Neurophysin I and anti-Neurophysin II, rabbit anti-c-Fos, anti-TRH, anti-A_2AR, anti-A₃R and anti-p-Creb1 (Ser 133), and mouse anti-β-Actin and anti-HA antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-Hu C/D and anti-Myc antibodies were purchased from Thermo Fisher (Waltham, MA) and Proteintech (Wuhan, China), respectively. Rabbit anti-Oxt antibody was obtained from Immunostar (Hudson, WI). Rabbit anti-A_2BR and anti-AVP antibodies were purchased from Bioss (Woburn, MA). Alexa Fluor 488/555 goat anti-rabbit, Alexa Fluor 488/555 goat anti-mouse, Alexa Fluor 488/555 donkey anti-rabbit and Alexa Fluor 488/555/633 donkey anti-goat secondary antibodies were also obtained from Thermo Fisher. Adenosine was obtained from Aladdin (Shanghai, China). Caffeine, CPA and Avertin was purchased from Amresco (Solon, OH), Tocris (Bristol, UK) and Sigma (St Louis, MO), respectively. Oxt was purchased from Sangon Biotech (Shanghai, China). OTR inhibitor L-368,899 was obtained from Santa Cruz.

Primary hippocampal cultures (6 or 23 DIV) were fixed with 4% PFA, permeabilized and blocked with a solution containing 0.2% Triton X-100, 10% goat serum, and 0.1% BSA in PBS. A rabbit polyclonal antibody anti-c-fos (Santa Cruz Biotechnology, Inc., sc-52; dilution 1:500), a mouse monoclonal antibody anti-CaMKII (Invitrogen, dilution 1:500), a rabbit polyclonal anti-MAP2 (Abcam, dilution 1:500) or a primary rabbit monoclonal anti-phospho-TrkA (Tyr674/675)/TrkB (Tyr706/707) antibody (Cell Signaling Technology; dilution 1:200) were diluted in blocking solution (10% goat serum and 0.1% BSA in PBS) and incubated at 4°C overnight. Secondary anti-rabbit or anti-mouse antibodies conjugated with Cy2, Cy3, or Cy5 (Jackson Immuno Research) were diluted 1:500 in PBS and incubated for 2 h at room temperature. Alexa Fluor 350 phalloidin (Lifetechnologies) was diluted 1:50 in PBS and incubated for 3 h at room temperature to selectively stain F-actin. Finally, cultures were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Biotium) diluted 1:1000 in PBS for 5 min, washed and mounted using an anti-fading aquous mounting medium (Fluoro-Gel Emsdiasum).

Mice were perfused with 4% paraformaldehyde in 0.1% phosphate buffer and post-fixed for 24 h in the same fixative. The head tissue, including the cochlea, OE, and olfactory bulb (OB), was decalcified with 10% EDTA solution, pH 7.0, and embedded in paraffin. Coronal sections were cut at 4 μm thickness and mounted on silane-coated slides. Deparaffinized sections were autoclaved at 121 °C for 20 min in Target Retrieval Solution (S1700; Dako) for antigen retrieval. Immunohistochemistry was performed with the following antibodies: anti-olfactory marker protein (OMP, goat polyclonal, 1:2000 dilution; Wako Chemicals), anti-NQO1 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-activated caspase-3 (rabbit polyclonal, 1:300; Cell Signaling Technology), anti-Ki67 (mouse monoclonal, 1:300; BD Biosciences), anti-c-fos (rabbit polyclonal, 1:50; Santa Cruz Biotechnology), anti-MnSOD (rabbit monoclonal, 1:100; Epitomics Inc.), and anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, goat polyclonal antibody, 1:100; Alpha Diagnostic International Inc.). Immunoreactivity was detected using the following antibodies in the Histofine Simple StainMAX-PO secondary antibody system (Nichirei) according to the manufacturers’ instructions, donkey anti-goat Alexa Fluor 488 (1:100; Invitrogen), and donkey anti-rabbit Alexa Fluor 594 (1:100; Invitrogen), incubated for 1 h at room temperature.

Methanol extract from the S. hexaphylla leaf was purchased from the Korean Plant Extract Bank (Daejeon, Korea). Recombinant soluble human M-CSF and RANKL were obtained from PeproTech EC Ltd. (London, UK). Monoclonal β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Anti-Akt, anti-phospho-Akt, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-phospho-IκB, and anti-phospho-PLCγ2 were purchased from Cell signaling Technology Inc. (Beverly, MA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fetal bovine serum (FBS), α-minimum essential medium (α-MEM), and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). All other chemicals were of analytical grade or complied with the standards required for cell culture experiments. Cyclohexamide (CHX), MG132, and Ac-Leu-Leu-nle-h (ALLN) were obtained from Calbiochem (San Diego, CA, USA).

Melatonin (N-acetyl-5-methoxytryptamine) and luzindole were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Recombinant RANKL and M-CSF were purchased from PeproTech (Rocky Hill, NJ, USA). Antibodies against phospho-ERK, ERK, phosphor-JNK, JNK, phosphor-p38, p38, phosphor-IκBα/β, and IκBα/β were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-NFATc1, anti-c-Fos, anti-RANK, anti-MT1 (MEL-1A-R; V-15), and anti-MT2 (MEL-1B-R; T-18) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The Leukocyte Acid Phosphatase (TRAP) kit and anti-β-actin antibody were obtained from Sigma-Aldrich.