Vdac1

Total RNA was extracted from cells using the TRIzol Reagent and purified with Direct-zol RNA kits (Invitrogen). cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen). The transcript cDNA was amplified by Real-time PCR using a Takara’s Perfect Real-Time PCR kit (Takara Bio, Otsu, Japan) and Applied Biosystems™ 7900 Real-Time PCR detection system (Thermo Fisher Scientific Inc., Waltham, MA, USA). All primers were purchased from Invitrogen, VDAC1 (F:5′-CGGGATCCATGATAAAACTTGATTTGAAAACG-3′, R:5′-GCGGCCGCTTATGCTTGAAATTCCAGTCC-3′) and β-actin (F:5′-CACGATGGAGGGGCCGGACTCATC-3′, R:5′-TAAAGACCTCTATGCCAACACAGT-3′). β-actin was used as an internal control to normalize sample differences.

Standard methods were used for the preparation and ligation of DNA fragments and for transformation and recovery of plasmid DNA from Escherichia coli [44 ]. Yeast was transformed by the LiAc procedure of Schiestl and Gietz [45 (link)]. Proteins were separated by SDS–PAGE [42 (link)]. Western blots were treated with rabbit polyclonal antibodies against anti F₁-β (a gift from Dr. Alexander Tzagoloff), anti-Atp6 (a gift from Dr. Jean-Paul di Rago and Dr. Marie-France Giraud), anti-HA (Biolegend, San Diego, CA, USA), and VDAC1 (Invitrogen, Waltham, MA, USA). The blots were then followed by a second reaction with peroxidase-coupled anti-rabbit IgG. The antibody complexes were visualized with the super signal chemiluminescent substrate kit (Pierce Chemical, Dallas, TX, USA). The protein concentrations were determined by the method of Lowry et al. [40 (link)].

Salts, buffers and Bovine serum albumin fraction V (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Colorimetric enzymatic kits were purchased from Sigma Aldrich (St. Louis, MO, USA) for fructose, GS Diagnostics SRL (Guidonia Montecelio, Rome, Italy) for glucose and uric acid and SGM Italia (Rome, Italy) for triglycerides. Elisa kit for insulin determination was purchased from Mercodia AB (Uppsala, Sweden). Chemiluminescent substrate, Immobilon (Millipore Corporation, Billerica, MA 01821, USA), Excellent Chemiluminescent detection Kit (ElabScience, Microtech, Naples, Italy), Polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and dye reagent for protein titration (Bio-Rad, Hercules, CA, USA) were used for Western blotting. The antibodies used for Western blot analysis were purchased by Cell Signaling (Danvers, MA, USA) for p-Akt and Akt1, Santa Cruz Biotechnology (Santa Cruz, CA, USA) for p-GSK, GSK-3β, GLUT-4 and VDAC1, Invitrogen (Carlsbad, CA, USA) for GLUT-5, Calbiochem (San Diego, CA, USA) for UCP-3, Abcam, (Cambridge, UK) for Oxphos, Biogenesis Ltd. (England, UK) for ANT, Sigma-Aldrich (St Louis, MO, USA) for actin.