Cd36 smφ

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 fixed by 4% paraformaldehyde 15 min, followed by permeabilization and blocking procedures. Cells were incubated with primary antibodies (1:100 dilution), CD36 (SMφ, Santa Cruz Biotechnology) x GFP (FL, Santa Cruz Biotechnology) and CAV-1 (N20, Santa Cruz Biotechnology) x hSTOM (E6, Santa Cruz Biotechnology) at 4 °C overnight. After washing cells by 1x Wash Buffer A, the cells were incubated with the PLUS and MINUS PLA probes for 1 h at 37 °C. Tap off the PLA probes solution and wash cells with Wash Buffer A twice. The ligation solution was applied and incubated with cells for 30 min at 37 °C. After the wash step, cells were incubated with the amplification solution for 100 min at 37 °C. Finally, wash cells two-time using 1x Wash Buffer B, followed by one-time 0.01x Wash Buffer B. The cells were mounted and analyzed in a confocal microscope.