Sirt5

Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (Thermo). Nuclear and cytoplasmic components were isolated using NE-PER nuclear and cytoplasmic extraction kit (Thermo). Homogenates (20 μg of total protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes.
Antibodies were used against the following proteins: Sirt6, Sirt1, Ac-H3K9, FoxO1, p-FoxO1 (Cell Signaling, Beverly, MA, USA), Ac-lysine, Sirt2, Sirt3 (Abcam, Cambridge, UK), ubiquitin (Santa Cruz Biochemicals, Dallas, TX, USA), Sirt4, lamin B, GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7, Ac-FoxO1 (LifeSpan Biosciences, Seattle, WA, USA), and HSP90 (Enzo Life Sciences, Plymouth Meeting, PA, USA).

Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (#78510 or 78505, Thermo Fisher Scientific, Waltham, MA, USA). Nuclear and cytoplasmic extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (#78835, Thermo Fisher Scientific). Homogenates (20 μg for Western blot and 200 μg for co-IP) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk, blots were probed with primary antibodies (Supplementary Table 3) against PAK4, p-PAK4 (S474), HSP90, ubiquitin, HMGCS2, phospho-PKA Substrate, phospho-(Ser/Thr), Sirt1, Sirt6, phospho-Akt (Ser473), Akt, acetylated-Lysine, CHOP, phospho-S6 Ribosomal Protein (Ser240/244), S6 Ribosomal Protein, NCoR1, NEDD4, ATF-6, phospho-PERK (Thr980) (Cell Signaling Technology), PAK4, FGF21, and PPARα (Santa Cruz Biotechnology), Sirt4, lamin B1, and GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7 and FoxO1 (Acetyl-Lys294) (LifeSpan Biosciences, Seattle, WA, USA), and Sirt2, Sirt3, GRP78 BiP, NCOR2/SMRT, CPT1α or CPT1A,T-OXPHOS, THRβ, p-IRE1α and MDM2 (Abcam, Cambridge, UK), LXRα (Proteintech, Rosemont, IL, USA). Immunoreactive bands were detected with an LAS-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).

Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (#78510 or 78505, Thermo Fisher Scientific, Waltham, MA, USA). Nuclear and cytoplasmic extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (#78835, Thermo Fisher Scientific). Homogenates (20 μg for Western blot and 200 μg for co-IP) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk, blots were probed with primary antibodies (Supplementary Table 3) against PAK4, p-PAK4 (S474), HSP90, ubiquitin, HMGCS2, phospho-PKA Substrate, phospho-(Ser/Thr), Sirt1, Sirt6, phospho-Akt (Ser473), Akt, acetylated-Lysine, CHOP, phospho-S6 Ribosomal Protein (Ser240/244), S6 Ribosomal Protein, NCoR1, NEDD4, ATF-6, phospho-PERK (Thr980) (Cell Signaling Technology), PAK4, FGF21, and PPARα (Santa Cruz Biotechnology), Sirt4, lamin B1, and GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7 and FoxO1 (Acetyl-Lys294) (LifeSpan Biosciences, Seattle, WA, USA), and Sirt2, Sirt3, GRP78 BiP, NCOR2/SMRT, CPT1α or CPT1A,T-OXPHOS, THRβ, p-IRE1α and MDM2 (Abcam, Cambridge, UK), LXRα (Proteintech, Rosemont, IL, USA). Immunoreactive bands were detected with an LAS-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).