Pparγ c26h12

Tissues were lysed in RIPA buffer as described in Müller et al., 2018 (link). Equal amounts of protein were separated by SDS-PAGE and transferred to a PDVF membrane. For protein detection, the following antibodies were used: C/EBPβ (E299, ab32358, 1:1000) from Abcam, C/EBPα (D56F10, #8178, 1:1000), PPARγ (C26H12, #2435, 1:1000), FAS (C20G5, #3180, 1:1000) and GAPDH (14C10, #2118, 1:1000) from Cell Signaling, SREBP1 2A4, MS-1207-PO, 1:1000 from NeoMarkers, and HRP-linked anti rabbit or mouse IgG from GE Healthcare. For detection, Lightning Plus ECL reagent (Perkin Elmer) or ECL prime reagent (GE Healthcare) was used. For re-probing, the membranes were incubated for 15 min with Restore Western Blot Stripping buffer (Thermo Fisher). The Image Quant LAS Mini 400 Imager or the Image Quant 800 Imager (both GE Healthcare) were used for detection and quantification of C/EBPβ LAP and LIP isoforms was performed using the supplied software.

Whole cell extracts were prepared by solubilizing cells in RIPA buffer supplemented with Complete Mini protease inhibitor cocktail (Roche Diagnostics; Indianapolis, IN, USA), followed by a freeze-and-thaw cycle, and clarification with centrifugation. Protein concentrations were quantified using the Micro BCA assay (ThermoFisher). Equal amounts of protein (25 μg) were resolved using 10% SDS-PAGE and immunoblotted according to standard protocols. The membranes were blocked with 5% non-fat milk prepared in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hr, followed by incubation with primary antibodies prepared in TBST at 4 °C overnight. Primary antibodies used in this study include: CD36 (SMφ) (Santa Cruz Biotechnology), ATGL (30A4) (Cell Signaling; Danvers, MA, USA), PPARγ (C26H12) (Cell Signaling), and phospho-β-catenin (D2F1) (Cell Signaling). Secondary antibody was peroxidase-conjugated goat anti-mouse or rabbit IgG (1:10000, Jackson ImmunoResearch; West Grove, PA, USA) prepared in TBST. The membranes were incubated at room temperature for 1 hr. Protein bands were visualized using Immobilon Western chemiluminescent HRP substrate (EMD Millipore; Billerica, MA, USA). The blots were stripped and reprobed with rabbit anti-GAPDH (FL-335) (Santa Cruz Biotechnology) to ensure equal loading.

Cells were lysed in loading buffer (Invitrogen, cat. # NP0007) containing protease inhibitor (Roche, cat. # 05892791001) and reducing reagent DTT (Invitrogen, cat. # NP0009), sonicated and subsequently boiled for 5 min. Approximately 20 μg of protein was loaded per lane and resolved by SDS polyacrylamide electrophoresis. Protein was transferred onto nitrocellulose membranes, blocked in 5% low-fat milk and probing was carried out overnight with antibodies to PPARγ (C26H12, 1:1000 dilution) (Cell signaling, cat. # 2435S), RXRα (D-20, 1:200 dilution) (Santa Cruz, cat. # SC-553), p65 (Santa Cruz, cat. # SC-8008, 1:1000 dilution), Vinculin (Sigma-Aldrich, cat. # V4505, 1:5000 dilution), and GAPDH (Sigma, cat. # G9545, 1:5000 dilution). Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Millipore, cat. # AP136P, 1:10,000 dilution) for 1 h and signal was developed using the enhanced chemiluminescence (ECL) method (GE Healthcare). Scans of the original western blots are shown in Supplementary Fig. 27.