Sirt5

Most chemicals including 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) were obtained from Sigma Aldrich (St. Louis, MO). Radio-labeled glucose and oleate were purchased from PERKin-Elmer NEN (Waltham, MA). Anti-actin, SIRT1, and JNK antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-SIRT3, SIRT5, Akt, phospho-Akt, AMPK, phospho-AMPK, ACC, phospho-ACC, phospho-JNK, insulin receptor, phospho-insulin receptor, acetylated lysine, PERK, phospho-PERK, Bip, p-38, phospho-p38, p65, phospho-p65, and IκBα antibodies were purchased from Cell Signaling Technology (Beverly, CA). Anti-F4/80 antibody was obtained from eBioscience (San Diego, CA). Anti-SIRT4 antibody was obtained from Abcam (Cambridge, UK).

Protein extracts were submitted to PAGE and transferred onto nitrocellulose membranes (32 (link)–34 (link)). Membranes were incubated with antibodies directed against SIRT5 (8782, 1:1,000, Cell Signaling Technology, Danvers, MA) or β-actin (4967S, 1:1,000, Cell Signaling Technology) and then with a secondary HRP-conjugated antibody (31460, 1:10,000, Thermo Fisher, Waltham, MA) (35 (link)). Blots were imaged with the ECL Western blotting system (GE Healthcare, Little Chalfont, UK). Images were recorded using a Fusion Fx system (Viber Lourmat, Collégien, France) (36 (link)).

Aliquots containing 40 μg of proteins from each lysate cells were subjected to SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories; Hercules, CA, USA) using Trans Blot Turbo Transfer System. Membranes were probed with the following primary antibodies: MitoProfile Total OXPHOS Human WB Antibody cocktail, Pyruvate Dehydrogenase E1-alpha (PDH) and pPDHSer²⁹³ (1:500; Abcam Cambridge, UK), Acetylated Lysine (1:1000 Novus, UK), pAKTSer⁴⁷³, AKT, NAMPT, SIRT1, SIRT3, SIRT5 (1:1000; Cell Signaling Technology) and β-ACTIN (1:5000; SIGMA Aldrich, St. Louis, MO, USA). After incubation with corresponding suited horseradish peroxidase-conjugated secondary antibody (1:2500; Cell Signaling Technology) signals were developed using the enhanced chemiluminescence kit (ClarityTM Western ECL Substrate, Bio-Rad) and the ChemiDoc Imaging System XRS + (BioRad) and analysed with the Image Lab 4.1 software. The intensity of bands corresponding to Mitoprofile, Acetylated Lysine, NAMPT, SIRT1, SIRT3 and SIRT5 was normalized to the β-ACTIN signal while phosphorylated AKT and PDH was normalized to total protein.

Resveratrol was purchased from SIGMA (Saint Louis, Missouri, USA). Radioactive labeled [7(N)-³H]-pregnenolone (12.6 Ci/mmol) and [1,2,6,7(N)-³H]–DHEA (63 Ci/mmol) were obtained from PerkinElmer (Waltham, MA, USA). [4-¹⁴C]-progesterone (ART-1398) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Antibodies against human CYP17A1 and POR were custom made by GenScript (Piscataway, NJ, USA). Anti-CYP21A2 antibody was a generous gift of Prof. Walter L. Miller (San Francisco UCSF, CA, USA). The phospho-Akt pathway was studied using anti-Akt and anti-phospho-Akt (Ser 473) antibodies from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SIRT1 (Abcam, Cambridge, UK), SIRT3 and SIRT5 (Cell Signaling, Danvers, MA, USA) were kindly shared by Dr. Jean-Marc Nouffer (University of Bern, Bern, Switzerland). β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). SIRT3/5 plasmids were obtained from the laboratory of Dr. Eric Verdin (University of California San Francisco, San Francisco, CA) via Addgene (Cambridge, MA) and provided by Prof Marion B. Sewer (UC San Diego, CA, USA). SIRT1 plasmid was obtained from Prof. Anna Biason-Lauber (University of Fribourg, Fribourg, Switzerland).

The primary antibodies used for immunohistochemistry, immunofluorescence, immunoprecipitation, and western blotting analysis were as follows: CDCP1 (Cell Signaling Technology #4115; Danvers, MA, USA), CDCP1 (Abcam #188818; Cambridge, UK), IdU (Abcam #181664), BrdU (Abcam #6326), HA (Abcam #18181), myc (Abcam #32072), Kras (Santa Cruz Biotechnology #sc-30; Dallas, TX, USA), Flag (Sigma-Aldrich #F1084), malonyl-lysine (PTM Biolabs 901; Chicago, IL, USA), succinyl-lysine (PTM Biolabs 401), glutaryl-lysine (PTM Biolabs 1151), acetyl-lysine (PTM Biolab 104), Sirt5 (Cell Signaling Technology 8782), tubulin (Abcam 7291), and TPI (Abcam #96696). The secondary antibodies used for immunofluorescence were Alexa Fluor 488 or 594 goat anti-mouse (#A-11001, #A-11005) or rabbit anti-mouse (#A-11034, R37117) IgG from Thermo Fisher Scientific (Waltham, MA, USA). The HRP-conjugated secondary antibodies (#7076, #7074, and #7077) used for western blot were from Cell Signaling Technologies.