Traf3

Cyclophosphamide (CPD) was purchased from Shengdi Company (Jiangsu, China). M-CSF and RANKL were purchased from Peprotech (New Jersey, USA). Primary antibodies against active β-catenin, Runx2, Cyclin D1, c-Myc, IκBα, phospho-IκBα, JNK, phospho-JNK, p38 and phospho-p38 were purchased from Cell Signaling Technology (Beverly, MA, USA) and others against TRAF-6, TRAF-3, NFATc1, RANK and c-Fos were purchased from Santa Cruz Biotechnology (CA, USA). The antibodies against β-catenin, phospho-Gsk-3β, and DKK1 were purchased from Abcam Company (Cambridge, UK).

Cells were lysed in Immunoprecipitation (IP) lysis buffer (40 mM Tris pH 7.5, 0.5% Triton-X-100, 100 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 2 mM Na₃VO₄, and EDTA-free cOmplete mini protease inhibitor cocktail (Roche, Basel, Switzerland)) on ice for 30 min. Lysates were pre-cleared for 10 min with protein G Dynabeads (Invitrogen). Antibody (Ab) recognizing one of the following was added: TRAF3 (Santa Cruz Biotechnology (Dallas, TX, USA) #sc-1828, 1:1000, or Cell Signaling Technologies (CST, Danvers, MA, USA) Ab #4729, 1:100) or GSK3β (CST, #12456). Lysates were incubated with IP Ab for 2 h at 4 °C, then Ab-bound targets were captured with protein G Dynabeads for 20 min at 4 °C. Dynabeads were washed 3X with IP washing buffer (20 mM Tris pH 7.5, 1% Triton-X-100, 40 mM NaCl) then resuspended in IP lysis buffer for Western blotting analysis.

After caerulein treatment, total protein samples were extracted from cells and separated by SDS-polyacrylamide gel electrophoresis. Next, the proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated with primary antibodies against TRAF3 (anti-rabbit, 1:500; Santa Cruz, Dallas, TX, USA), Bcl-2 (anti-rabbit, 1:500), C-caspase 3 (anti-rabbit, 1:500), Bax (anti-rabbit, 1:500), p-p38 (anti-rabbit, 1:500), p38 (anti-rabbit, 1:500), and GAPDH (anti-mouse, 1:1,000; Santa Cruz) at 4°C overnight. After washing with TBST, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution; Santa Cruz Biotechnology Inc.) for 1 h at room temperature. The blots were measured using a Pierce ECL Plus Substrate (Thermo Scientific, USA) according to the manufacturer’s instructions.