Western Blotting

mBMMs (1.0 × 10⁵ cells/well) were harvested from a 12-well plate and treated with RANKL (100 ng/ml) and M-CSF (100 ng/ml) in the presence and absence of TER (10 μM) for 10–30 min (to detect PKCα, phospho-PKCα/βII, and phospho-PKCδ expression). After each time course, cells were lysed rapidly by adding ice-cold cell lysis buffer containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.2), 1% Nonidet P-40, 5 mM EDTA-Na, 1 mM sodium ortho vanadate, 1% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail (Sigma-Aldrich) for 10 min, according to a previously described method (Omori et al., 2004). Protein concentration was determined using a Bradford assay by using bovine serum albumin (BSA, Sigma-Aldrich) as the standard (Bradford, 1976). The lysates (30 μg) were mixed with SDS sample buffer (1% [w/v] SDS, 45 mM Tris-HCl [pH 6.8], 15% [v/v] glycerol, 144 mM 2-mercaptoethanol, and 0.002% bromophenol blue), and samples were boiled for 5 min. The samples were separated using SDS-polyacrylamide gel electrophoresis (PAGE) on a 7.5% or 12% polyacrylamide slab gel and transferred onto polyvinylidene difluoride membranes. These membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20® (TBST; 20 mM Tris-HCl (pH 7.6) containing 150 mM NaCl and 0.1% (v/v) Tween 20®) for 1 h and incubated with anti–phospho-PKCα/βII (1:1,000) and anti-total PKCα (1:1,000) overnight at 4°C. The membranes were incubated with secondary antibodies (goat anti-rabbit immunoglobulin G (IgG)–horseradish peroxidase (HRP) conjugate, 1:2,000 dilution; GE Healthcare, Chicago, IL, United States) for 1 h. HRP activity was visualized by incubating the membranes and using an electrochemiluminescence (ECL) detection system (SuperSigna® West Dura Extended Duration Substrate, Thermo Fisher Scientific), followed by autoradiography. At the end of these experiments, the immunodetection system and the bound antibody were removed from the blots by incubating the membranes with re-probing buffer (Restore™ Western Blot Stripping Buffer, Thermo Fisher Scientific). The blots were then stained with anti–β-actin antibody (1:2,000 dilution) to confirm that equal amounts of protein were present in each lane of the gel.