Huwe1

Unless otherwise specified, cells were lysed in buffer composed of 50 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton, 0.2 mM EDTA, 5% glycerol, and 1 mM PSMF. Cell lysis was achieved by vortexing on ice followed by sonication using Bioruptor (Diagenode) for 10 cycles of 30 s on, 30 s off. Lysates were cleared by centrifugation for 10–15 min at 21,000g and 4°C. Protein concentration was assessed using a BCA assay (Thermo Fisher Scientific) and then equalized across samples, typically to a final concentration of 1–2 mg/ml. NuPAGE 4–12% Bis–Tris gradient gels were used for all immunoblots (Thermo Fisher Scientific). Imaging was performed with Bio-Rad ChemiDoc Touch Imaging System (732BR0783) after incubation in HRP substrate (Immobilon, Millipore). Blots were analyzed using ImageLab v6.0.1 (Bio-Rad). The antibodies used were as follows (name, catalog number, dilution): HAPSTRl (OTl2B8; 1:1,000; OriGene), FLAG (F3165; 1:5,000; Sigma-Aldrich), HA (26183; 1:5,000; Thermo Fisher Scientific), HUWE1 (ab70161; 1:1,000; Abcam), vinculin (V9131; 1:10,000; Sigma-Aldrich), HO-1/HMOXl (NBPl-97507; 1:1,000; Novus), p21/CDKN1A (2947; 1:1,000; CST), p53/TP53 (P6749; 1:2,000; Sigma-Aldrich), HRP anti-rabbit IgG secondary (7074; 1:10,000; CSF), HRP anti-mouse IgG (31430; 1:10,000; Thermo Fisher Scientific).

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).