Protein a g sepharose beads

After the treatment with metformin (2 mM), glucose (15 mM) and/or siAMPK (50 nM)/siControl (50 nM) or Compound C (1 μM) at 24 h or 48 h, the cells were lysed in ice-cold NP-40 buffer containing protease inhibitor (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). The whole-cell lysates were incubated with protein A/G Sepharose beads (Roche) for 2 h at 4 °C, and the beads were discarded to eliminate non-specific binding. Then, the supernatants were incubated overnight with a Bcl-xl antibody (Cell Signaling Technology, Danvers, USA) at 4 °C, followed by incubation with protein A/G Sepharose beads for another 2 h at 4 °C. Then, western blotting was performed with the indicated antibodies according to standard protocols as previously described^{20 (link)}. The primary antibodies, including p-AMPK (T172) (#50081), phospho-Acetyl-CoA carboxylase (p-ACC) (#11818), phospho-Tuberin/TSC2 (p-TSC2) (Ser1387) (#23402), phospho-Raptor (p-Raptor) (Ser792) (#2083), p-mTOR (S2448) (#5536), phospho-p70 S6 kinase (p-S6K1) (Thr389) (#97596), p-ULK1 (Ser757) (#14202), p-4E-BP1 (Thr70) (#9455), cleaved Caspase-3 (Cl-Caspase3) (Asp175) (#9664), p-Akt (Ser473) (#4060), p-Akt (Thr308) (#13038), RagB (#8150), LC3B (#3868), β-Actin (#3700) were all purchased from Cell Signaling Technology, Danvers, USA.

BMMs were pre‐treated without or with Nepetin (6.25 μmol/L) for 2 hours followed by treatment with RANKL (50 ng/mL) for 30 minutes. After RANKL stimulation, cells were lysed by Western and IP lysis buffer supplemented with PMSF protease inhibitor for 30 minutes. Cell lysates were cleared by centrifugation, and 500 μg of total protein was treated with anti‐TRAF6 (5 μg) or anti‐Beclin 1 (5 μg) antibodies overnight at 4°C under constant and gentle rotation. Total protein incubated with non‐specific IgG antibody was applied as a negative control. Subsequently, 20 μL of protein A/G‐sepharose beads (Cell Signaling Technology) was added to each sample and then incubated at 4°C overnight under constant and gentle rotation. The beads were denatured by boiling in 2× SDS loading buffer for 5 minutes. After centrifugation, proteins were resolved on 10% SDS‐PAGE gels.