Immunoblotting protocol for protein analysis

Immunoblotting was performed as previously described [44 (link)]. The cells were grown in a 12-well plate, and the experiments were repeated at least three times. Cell lysates were prepared using a cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1 μM PMSF (Roche, Basel, Switzerland). The protein levels of the obtained lysates were quantified using BCA Protein Assay reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cell lysates were diluted in a 5× sodium dodecyl sulfate (SDS) sample buffer and then separated on 10% SDS-PAGE gel. The separated proteins were transferred onto polyvinylidene fluoride membranes (Invitrogen, Carlsbad, CA, USA), which were then incubated with appropriate primary and secondary antibodies. Specific proteins were detected using rabbit anti-PPARγ (Cell Signaling Technology), mouse anti-SREBP-1 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CREB (Cell Signaling Technology), rabbit anti-phospho-CREB (ser133, Cell Signaling Technology), chicken anti-ADRB2 (Abcam, Cambridge, MA, USA), and mouse anti-GAPDH (Santa Cruz Biotechnology). Protein bands were detected using horseradish peroxidase-conjugated anti-chicken IgY (Abcam), anti-rabbit IgG (AbFrontier, Seoul, Korea), and anti-mouse IgG (AbFrontier) antibodies, and enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).