Orthovanadate

Cells were collected by centrifugation, washed twice in ice-cold PBS and lysed in ice-cold RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/mL PMSF, 40 mg of aprotinin/mL, 100 mM orthovanadate) (all reagents from Sigma-Aldrich). After centrifugation (15000 g, 15 min, 4 °C) to separate cellular debris, the lysates were resolved in a 12% SDS-PAGE, and electrotransferred onto a nitrocellulose paper (Protran; Schleicher and Schuell, Dassel, Germany). Determination of protein levels was carried out by immunoblot analysis using antibodies against p53 (CM5; Leica Biosystems, Barcelona, Spain), p-p53^Ser15 (9284; Werfen), p-H2AX Ser139 (07–164, EMD Millipore, Madrid, Spain), p19ARF (ab80; Abcam), p16 (M-156; Santa Cruz Biotechnology, Heidelberg, Germany), pan-Ras-V12 (Ab-1; Sigma-Aldrich) and β-tubulin (T5168; Sigma-Aldrich).

Cell lysates were obtained using modified radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Complete, Boehringer Mannheim, Mannheim, Germany), NaF (50 mM), PMSF (1 mM), aprotinin, leupeptin, pepstatin (1 ng/ml) and orthovanadate (2 mM) (Sigma-Aldrich). The following primary antibodies were used for immunodetection: rabbit anti-total AKT, anti-phospho-AKT (Thr 308) and anti-phospho AKT (Ser 473) and used at 1:1000 dilution (cell signaling), rabbit anti-total Erk1/2 (dilution 1:2000) and rabbit anti-phospho-Erk1/2 (Thr202/Tyr204) (dilution 1:1000) (cell signaling), mouse anti β-actin used at 1:5000 dilution (Sigma-Aldrich) and rabbit anti-vinculin used at 1:30000 dilution (Abcam). Goat anti-rabbit IgG HRP-linked (cell signaling) and goat anti-mouse IgG HRP-conjugate (Bio-Rad) were used as secondary antibodies at 1:2000 dilution.

Aprotinin, cisplatin (cis-diamminedichloroplatinum(II)), p-coumaric acid, ethanol, N, N-dimethylformamide (DMF), luminol, orthovanadate, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma-Aldrich (Taufkirchen, Germany). Dulbecco’s modified Eagle’s medium (DMEM) with 4 mM L-glutamine and 4.5 mg/ml glucose was obtained from Lonza (Cologne, Germany). Fetal calf serum (FCS) and phosphate-buffered saline (PBS) were purchased from PAN Biotech (Aidenbach, Germany). Leupeptin was purchased from Biomol (Hamburg, Germany). Dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), glycerol, hydrogen peroxide (H₂O₂), sodium chloride (NaCl), Tris hydrochloride (Tris-HCl), and Tris ultrapure were purchased from AppliChem (Darmstadt, Germany). PD98059 was purchased from Tocris Bioscience (Wiesbaden-Nordenstadt, Germany), and penicillin-streptomycin was bought from Invitrogen (Darmstadt, Germany). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was obtained from Ferak (Berlin, Germany), and SB203580 was purchased from Enzo Life Sciences (Lörrach, Germany). Triton^® X-100 was bought from Roth (Karlsruhe, Germany).

Tetracycline, oxyTetracycline, chlorTetracycline, doxycycline, ethidium bromide (EB), Hoechst33342 stain, cefoperazone, cefazolin, streptomycin, ampicillin, roxithromycin, chloramphenicol, rifampicin, norfloxacin, deoxycholate, sodium cholate, ofloxacin, doxorubicin, daunorubicin, acridine flavin, and quinine were purchased from Coolaber (Beijing, China), and ortho-vanadate was purchased from Sigma-Aldrich (St. Louis, MO, United States). Carbonylcyanide m-chlorophenylhydrazone (CCCP) and reserpine were obtained from MedChemexpress (Monmouth Junction, NJ, United States). Drug-sensitive paper was purchased from Hangzhou Microbial Preparation (Hangzhou, China).

Total protein was extracted from adipocytes and stromal vascular fraction (SVF) isolated from SAT, and in vitro differentiated adipocytes on day 7 and 14 of differentiation. Cells were lysed in ice-cold lysis buffer: 25 mM Tris-HCl (Sigma), pH 7.4; 0.5 mM EGTA (Sigma); 25 mM NaCl (Sigma); 1% Nonidet P-40; 10 mM NaF; 100 nM okadaic acid (Alexis Biochemicals), 1X Complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and 1 mM orthovanadate (Sigma). Lysates were vortexed and incubated on ice for 10 minutes and then centrifuged at 15,000 g for 15 minutes at 4°C. The infranatant was transferred into a new tube and saved at −80°C. Protein concentration was determined using a BCA protein assay kit (Pierce, Thermo Scientific). 10 µg proteins were separated by SDS-PAGE (5–8% gradient stain-free gels; BioRad), transferred to nitrocellulose membranes and blocked with 0.05% tween-PBS with 5% BSA. Membranes were incubated overnight with the primary antibody anti-CABLES1 (1:240, HPA073649; Sigma-Aldrich). Membranes were washed with 0.05% tween-PBS and incubated for 1 hour at room temperature with horseradish peroxide-conjugated secondary antibody (anti-rabbit, 1:2000; Cell Signalling). Protein bands and stain-free blot images were detected using enhanced chemiluminescence and the ChemiDocTM MP System (Biorad) and quantified using the Image Lab Software (Version 6.1.0; BioRad).

Sodium fluoride and orthovanadate, phenylmethylsulfonyl fluoride, aprotinin and leupeptin were purchased from Sigma (Saint-Louis, MO, USA). Anti-ERK1/2, anti-phospho-ERK1/2 and HRP conjugated anti-rabbit antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-Hsp90 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-CD19 and anti-CD103 conjugated antibodies were purchased from miltenyi (Bergisch Gladbach, Germany). HRP-conjugated anti-mouse antibody was from Dakopatts (Glostrup, Denmark).