Protein g agarose slurry

LN229 cells were transiently transfected with siRNAs for 48 h, then washed with cold PBS and lysed in culture dishes with IP buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate, and 1 mM EDTA) supplemented with protease and phosphatase inhibitors for 15 min on ice [29 (link)]. Cell lysates were scraped off dishes and briefly sonicated and their protein concentration measured via BCA assay (23225, Thermo Fisher Scientific). An amount of 500 μg of whole-cell extract proteins were incubated with antibodies against DR5 or integrin β1 (1 μg/mL) overnight at 4 °C, then 30 μL prewashed Protein G agarose slurry (sc-2002, Santa Cruz Biotechnology, Dallas, TX, USA) was added, and the mix was incubated for 2 h at 4 °C under mild rocking. Bead-bound protein complexes were washed three times with IP buffer and eluted with 2× Laemmli sample buffer by boiling for 5 min. Immunoprecipitated proteins were separated by SDS-PAGE and detected via immunoblotting.

U2OS and MG63 cells (1 × 10⁵ per well) were seeded in 6-well plates and treated with vehicle control or metformin. Cells were harvested and lysed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA). Culture media from the treated cells were condensed with a Microcon YM-10 (Millipore, Bedford, MA, USA) for Ang1 Western blotting analysis. Protein concentrations were determined with a Bradford assay (Bio-Rad). Each protein sample was separated on 10 or 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore). After blocking for 1 h with a buffer containing 0.05% Tween20 and 3% bovine serum albumin (Sigma), the membrane was probed with specific antibodies and incubated overnight at 4°C. Primary antibodies against p-p38 MAPK, p38 MAPK, ALP, COL1A1, BSP, OC (Cell Signaling Technology), RUNX2, OSX, Dlx5, β-actin, IL-6, p-Iκbα, Iκbα, IKKβ, NFκB p65, c-Src, TRAP, Cathepsin K (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Ang1, and Ang2 (Abcam) were used at a 1:1000 dilution. For immunoprecipitation analysis, cells were washed twice with PBS and lysed with RIPA buffer. The lysates were centrifuged and incubated with an antibody, followed by addition of protein A- or protein G-agarose slurry (Santa Cruz Biotechnology). Protein A/G beads were collected, washed, and resolved by 10 or 12% SDS-PAGE and analyzed by Western blotting.