Western Blotting and Immunoprecipitation Protocols

For IB experiments, mouse liver tissues and cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 8.0], 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na₃VO₄, and 1X protease inhibitor cocktail (Sigma, St. Louis, MO). Proteins were resolved on 6% or 8% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membrane was blocked for 1 h at room temperature in 10% skim milk solution and incubated with several primary antibodies such as anti-BMAL1 [20 (link)], CLOCK (SantaCruz, Dallas, TX), β-GAL (Promega, Madison, WI), MYC (SantaCruz, Dallas, TX), ACTIN (SantaCruz, Dallas, TX). Immunoreactive bands were visualized with ECL reagents (Thermo Scientific, Waltham, MA) according to manufacturer's instructions. For IP experiments, NIH3T3 cells were transfected with the indicated plasmids. At 36 hours posttransfection, the cells were lysed with RIPA buffer and centrifuged at maximum speed for 20 min at 4°C. Equal amounts of total protein were incubated with 2μg of anti-Flag M2 (Sigma, St. Louis, MO) for 1.5 h at 4°C and then added protein A/G-Sepharose bead slurry. The final immune complexes were analyzed by immunoblotting with indicated antibodies [21 (link)].