To prepare extracts, tissues were collected from male C57BL/6 mice and suspended in lysis buffer (51) [50 mM HEPES-KOH (pH 7.5), 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, 10 mM NaF, 0.25 mM Na3VO4, and 50 mM β-glycerolphosphate] supplemented with cOmplete Protease Inhibitor (Roche #04693116001). After homogenization, the cell extracts were centrifuged at 20,000g for 20 min at 4°C. The supernatant extracts were used for immunoprecipitation and Western blots. Equal amounts of protein were electrophoresed on 10% SDS–polyacrylamide gels, and the bands were transferred to polyvinylidene fluoride membranes (Millipore, USA). Immunoreactive bands were detected and analyzed with a Bio-Rad ChemiDoc MP imaging System and Image Lab Software (Bio-Rad, USA). Relative protein levels in each sample were normalized to β-Actin to standardize the loading variations. The primary antibodies for immunoblotting included anti–β-Actin (1:10,000 dilution; Cell Signaling Technology, #4970) and anti-ZCWPW1 (1:1000 dilution; produced as described above).

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