Ab2739

For western blotting analysis, TamS and TamR cells were seeded into 6-well plates, using 1.8 × 10⁵ cells per well in their respective media. Cells were treated with increasing concentrations of ST045849 (obtained from TimTec) and OSMI-1 (gift from Professor Suzanne Walker, Harvard Medical School, or obtained from Sigma Aldrich) the next day and collected on ice 24 h after the start of the treatment. Immunoblotting was performed using RL2 antibody (for total O-GlcNAcylation) (Abcam, ab2739), OGT (Cell Signalling, #5368) and β-actin antibody (Cell Signalling, #4967). Band intensity was quantified using ImageJ software.

α-O-GlcNAc (RL2, Abcam, ab2739, 1:1000; CTD110.6, Cell Signaling, #12938, 1:10,000), α-OGT (Cell Signaling, #5368, 1:1000), α-SCD1 (ThermoFisher, PA5-17409, 1:1000), α-SCD2 (G15, Santa Cruz, sc-14722, 1:200), α-PPARγ (D69, Cell Signaling, 2430, 1:1000), α-phospho-PPARγ (AW504, Millipore, 04-816, 1:1000), α-Acetyl CoA carboxylase 1 (Cell Signaling, 4190, 1:1000), α-Fatty acid synthase (Novus, NB400-114, 1:1000), α-Nape-pld (Abcam, ab95397, 1:1000), α-FAAH (Abcam, ab54615, 1:1000), α-FLAG (M2, Sigma, A8592, 1:10,000), α-GAPDH (FL-335, Santa Cruz, sc-25778, 1:1000), and α-alpha-Tubulin (B-5-1-2, Sigma, T5168, 1:1000) were purchased from vendors. Tissues were lysed in buffer containing 1% Nonidet P-40, 50 mM Tris-HCl, 0.1 mM EDTA, 150 mM NaCl, proteinase inhibitors and protein phosphatase inhibitors. Thirty micrograms of protein lysate were electrophoresed on SDS-PAGE gels and transferred to methanol-activated PVDF membranes. Primary antibodies were incubated at 4 °C over the night. Western blotting was visualized by peroxidase conjugated secondary antibodies and ECL chemiluminescent substrate. Uncropped western blots images were shown in Supplementary Fig. 12 and Suplementery Fig. 17. O-GlcNAc signals detected by CTD110.6 antibody were visualized by SuperSignal ECL (ThermoFisher, 34095). Experiments were repeated three times.

To determine differences in post-translational modifications (PTMs), each variant was immunoprecipitated from mammalian cells. HEK293T cells were grown on 6-well plates, co-transfected with or without OGT and either Pgc1α WT or Pgc1α p.C430S. When specified, cells were incubated in DMEM supplemented with PUGNAc at 100 μ3ϕoρ 18η to inhibit O-GlcNAcase. Cells were lysed in 50 mM TRIS, pH 8, 150 mM NaCl, 1% NP-40, 0.1 mM EDTA, 2 mM nicotinamide, 10 mM NaF and protease inhibitor cocktail. Clarified cell lysates were incubated with 5 μg of anti-myc (Invitrogen 9E10 clone) at 4^°C overnight. Pgc1α was immunoprecipitated using 25 μL of Dynabeads (Invitrogen) at 4^°C for 2 hours. Non-transfected cells were used as control. Samples were boiled in SDS-PAGE loading buffer, run in 8% gels and transferred to PVDF membranes. These membranes were incubated with anti-myc, anti-O-GlcNAc (Abcam, Ab2739, 1:5000), anti-acetyl-lysine (Cell Signal, 9441, 1:1000), anti-PhosphoSer/Thr (Abcam, Ab17464, 1:1000) and anti-PPARγ (Santa Cruz, sc-7196, 1:500). Clean blot HRP detection kit (Thermo Fisher Scientific) was used to detect PPARγ from immunoprecipitates. Band density was analyzed using ImageJ.