Anti c fos

Protein concentrations (n = 4) were determined in the supernatant of liver tissues by classic BCA protein assay (Beyotime). Equal amounts of protein from each sample were fractionated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes using Bio-Rad western blotting apparatus. The membranes were blocked with 5% fat-free milk or 5% bovine serum albumin, and then probed with the following primary antibodies for 12 h at 4°C: anti-GAPDH (1:2000), anti-PI3K (1:1000), anti-Akt (1:1000), anti-p-Akt (1:1000), anti-PTEN (1:500), anti-Raptor (1:1000), anti-Rictor (1:800), anti-p-Rb (1:1000), anti-C-fos (1:800), anti-C-myc (1:1000), anti-C-jun (1:1000), anti-Fas (1:1000), and anti-Fas-L (1:1000) (Abcam, Cambridge, United Kingdom). The membranes were incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (1:2000–1:3000, Abcam), and visualized with an enhanced chemiluminescence (ECL) detection kit (Millipore). Bands were quantified using Quantity One version 4.40 software (Bio-Rad).

Cells were grown in 60 mm dishes and treated with 3 Gy radiation, shRNA2, or a combination of both radiation and shRNA2. Cells were washed with ice-cold PBS and scraped into ice-cold lysis buffer (comprised of TRIS-HCl pH 7.8 20 mM, NaCl 137 mM, EDTA pH 8.0 2 mM, NP40 1%, glycerol 10%, NaF 10 mM, Leupeptin 10μg/mL, Na2VO4 200 μmol/L, PMSF 5mM, and Aprotinin (Sigma-Aldrich, MO, USA). Lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4°C, and supernatants removed and assayed for protein concentration using the Pierce BCA bovine serum albumin. Protein was quantified using BCA protein assay (Thermo Scientific), separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF;Bio-Rad), and probed with the indicated antibodies. Bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific). Anti- c-Fos was purchased from Abcam; Anti-cyclinB1, Anti-cleaved PARP and anti-GAPDH were purchased from Cell Signaling Technology. Donkey-anti-rabbit and sheep-anti-mouse horseradish peroxidase-conjugated secondary antibodies were purchased from GE Healthcare. Images were captured with a FUJIFILM LASS-3000 camera system.

Taxifolin (HPLC ≥ 98%) was purchased from Sigma-Aldrich (Shanghai, China). Recombinant soluble human M-CSF and mouse receptor activator of nuclear factor-κ B ligand (RANKL) were obtained from PeproTech (Rocky Hill, NJ, United States). The MTT Cell Proliferation and Cytotoxicity Assay Kit was purchased from Boster (Wuhan, China). The following antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States): ERK (#9102), phospho-ERK (#4377), JNK (#9258), phospho-JNK (#4668), P38 (#8690), phospho-P38 (#4511), IKKβ (#8943), phospho-IKKα/β (#2697), P65 (#8242), phospho-P65 (#3033), IκB-α (#4812), phospho-IκB-α (#2859), NFATc1 (#8032), and RANK (#4845). Anti- C-Fos was purchased from Abcam (Cambridge, MA, United States). Antibodies against Cathepsin K, MMP-9, and TRAP were obtained from Proteintech Group (Wuhan, Hubei, China) The NF-κB and AP-1 probe was purchased from Beyotime (Shanghai, China). The TRAP staining kit and all other reagents were purchased from Sigma-Aldrich.

The celiac ganglia were dissected from MeAàVMH^hM3Dq or mCherry control mice euthanized 2h after CNO administration. The tissue was post-fixed in 4% paraformaldehyde at 4°C overnight, cryo-protected in 30% sucrose (Sigma-Aldrich, 50389) in PBS, embedded in O.C.T Compound (Thermofisher Scientific, Watham, MA; 23–730-572), frozen at −80°C, and sectioned at 10μm thickness. Slides were washed in 0.03% PBT (3 × 5 minutes), incubated in blocking solution overnight at 4°C (2% normal donkey serum, 3% bovine serum albumin in 0.03% PBT), incubated in primary antibodies for 48h at 4°C (Cell Signaling anti-cfos – 1:100; abcam chicken polyclonal to tyrosine hydroxylase [#ab76442] – 1:500), washed in 0.03% PBT (3 × 5 minutes), incubated in secondary antibodies for 2h at RT (Jackson AF-647 donkey anti-rabbit – 1:250; Jackson AF-594 donkey anti-chicken – 1:500), and washed in 0.03% PBT (3 × 5 minutes). After staining, tissue sections were mounted with DAPI counterstain (Fluoromount).