Immunoprecipitation and Western Blot Analysis

Cells were lysed on ice for 20 min in a buffer containing 1% Triton X-100, 10 mM Tris (pH 7.6), 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 mM EDTA, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche). The resulting lysates were centrifuged at 16,000 × g at 4 °C, and the supernatant fraction was incubated with antibodies against FLAG (Cell Signaling Technology), and HDAC6 (Cell Signaling Technology) for 2 h at 4 °C, followed by incubation with Protein G Plus Agarose (Merck Millipore). Immunoprecipitants were washed two times with the lysis buffer, boiled in 5× SDS loading buffer for 10 min, followed by separation on SDS-PAGE and analysis by western blot. Western blot analysis was performed as described above. For inflammatory IL-1β-induced protein interaction, WT or Parkin^−/− OCPs were cultured for 3 days with 10 ng/ml of IL-1β in the presence of RANKL and M-CSF. Then, the anti-HDAC6 immunoprecipitation was performed.