Fabp4

SDS-PAGE and immunoblot analysis of samples were performed using the following antibodies: custom-made PDGF D (22 (link)), fatty acid synthase (Cell Signaling Technologies [CST]; catalog no.: 3180), C/EBPα (CST; catalog no.: 8178), peroxisome proliferator–activated receptor gamma (CST; catalog no.: 2435), perilipin (CST; catalog no.: 9349), FABP4 (CST; catalog no.: 2120), acetyl CoA carboxylase (CST; catalog no.: 3676), β-actin (CST; catalog no.: 4970), p-AKT (CST; catalog no.: 9275), AKT (CST; catalog no.: 9272), p-JNK (CST; catalog no.: 4668), JNK (CST; catalog no.: 9258), p-ERK1/2 (CST; catalog no.: 9101), ERK1/2 (CST; catalog no.: 9102), p-β-PDGFR Y751 (CST; catalog no.: 3161), p-β-PDGFR Y771 (CST; catalog no.: 3173), β-PDGFR (CST; catalog no.: 3169), vinculin (Sigma; catalog no.: V9131), ubiquitin (CST; catalog no.: 3936), ubiquitin-K48 (Abcam; catalog no.: ab140601), ubiquitin-K63 (Abcam; catalog no.: ab179434), HUWE1 (Abcam; catalog no.: ab70161), and streptavidin–horseradish peroxidase (Abcam; catalog no.: ab7403).

Protein lysates from tumor tissues or cultured cells were prepared using modified RIPA buffer containing proteinase and phosphatase inhibitors. Protein concentrations were determined using the BCA protein assay reagent kit (Pierce Biotechnology, Rockford, IL, USA). Lysates were loaded and separated on SDS-polyacrylamide gels. The proteins were then transferred to a nitrocellulose membrane. The membrane was blocked at room temperature for 1 h in 5% milk powder in Tris-buffered saline with Tween 20 (TBST) and then incubated at 4 °C overnight with primary antibodies: FABP4 (catalog number HPA002188; SigmaAldrich) and vinculin (catalog number V9131; Sigma) or beta-actin (catalog number A5316, Sigma) as loading control. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (for FABP4) or sheep anti-mouse IgG (for vinculin) (catalog numbers NA934 and NA931; 1:2000; GE Healthcare) at room temperature for 2 h. An enhanced chemiluminescence detection kit (catalog number NEL104001EA; Pierce Biotechnology) was used to visualize the horseradish peroxidase signal. Full blots are included in Supplementary Fig. 8.

Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).