α actin

CPD and 6-4PP quantitation was performed using commercial ELISA kits (CosmoBio, Carlsbad, CA, United States) according to the manufacturer’s instructions. Antibodies used in this study include anti-DDB2 (#5416), FLAG (DYKDDDDK, #2368), histone H2B (#5546), α-actin (#4970) (Cell Signaling Inc. Danvers, MA, United States), HA (3F10, Millipore-Sigma, St. Louis, MO, United States), V5 (Bethyl Laboratories, Montgomery, TX, United States), CPD (clone TDM-2; CosmoBio) XPC (graciously supplied by Jeff Salisbury) (Acu et al., 2010 (link)).

Western blotting was conducted as previously described^{33 (link),37 (link),41 (link),52 (link),53 (link)}. The following primary antibodies were used in this study: α-actin (Cell signaling #4976L), SLC25A1 (Proteintech #15235-1-AP), SLC13A5 (Santa Cruz #sc293277), AT-1 (Aviva System Biology #ARP43888_P050), Acetylated Lysine (Cell Signaling #cs9441L), ACLY (Abcam #ab40793), Calnexin (Novus #NB100-1974). Donkey anti-rabbit or goat anti-mouse IRDye 800CW and 680RD-conjugated secondary antibodies (LI-COR Biosciences, #926-32213, #926-32210, #926-68073, #926-68070) were used for infrared imaging (LI-COR Odyssey Infrared Imaging System; LI-COR Biosciences). For enriched liver ER and nuclear Western blotting, target proteins were normalized to total protein staining with the Revert Total Protein Stain (LI-COR Biosciences, #926-11021) performed before immunodetection. Original uncropped Western blot images included in the manuscript can be found in Supplementary Fig. 9.

Glutathione S-transferase (GST)-fused proteins (PPARγ domain mutants) immobilized with glutathione-agarose were incubated with TRIM25-expressing cell lysates for 2 h at 4 °C. Protein complexes were pulled down by centrifugation and washed four times with binding buffer. Precipitates were detected by immunoblotting using anti-GST or TRIM25 antibodies. For analyzing interactions between endogenous PPARγ and TRIM25, 3T3-L1 adipocytes were lysed with binding buffer. Cell lysates were incubated with anti-PPARγ or TRIM25 antibodies and analyzed by western blotting. HEK-293 cells expressing PPARγ, TRIM25, or their mutants were lysed in binding buffer, and total cell lysates were incubated with an anti-hemagglutinin (HA) antibody at 4 °C. Immunoprecipitants or total cell lysates were analyzed with specific antibodies as indicated. The antibodies used in this study included α-TRIM25, α-PPARγ, α-GST, α-Ub, α-aP2, and α-adipsin antibodies, which were purchased from Santa Cruz Biotechnology (Dallas, TX), while α-HA, α-actin, α-HSP90, and α-adiponectin were purchased from Cell Signaling Technology (Danvers, MA).

Denuded aortae were pulverized in liquid nitrogen, re-suspended in ice-cold cell lysis buffer (50 mM Tris [pH 7.4], 50 mM NaF, 1 mM Na₄PPi, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, and 1% cocktail of protease inhibitors) and protein concentrations determined using Coomassie Plus Protein Assay Reagent (Perbio, USA). Homogenates were run at several protein concentrations (2.5, 5 and 7.5 µg/lane) on NuPAGE Novex 4–12% Bis-Tris mini gels (Life Technologies) and transferred to a nitrocellulose membrane, and IP3R was detected using rabbit polyclonal anti-IP3R1 (No. 3763 used at 1:1,000; Cell Signaling Technology). Large conductance calcium-activated potassium channels (BK_Ca) in lysates were detected using an anti BK_Ca mouse monoclonal antibody (1:500 dilution, ab99046; ABcam). Secondary HRP-conjugated antibodies were either goat anti-rabbit HRP conjugated for IP3 detection (ab6721; Abcam) or rabbit anti-mouse HRP conjugated for BK_Ca detection (ab6728; Abcam). IP3R and BK_Ca protein abundance was quantified using Quantity One software (BioRad). Expression was normalized to GAPDH (1:40,000. No. G8795; Sigma-Aldrich) or α-actin (1:1,000, No. 14958; Cell Signaling Technology).

The heart tissues were homogenized with cold lysis buffer. Western blotting was performed as previously described^{49 (link)}. The homogenates were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blotting, the membranes were probed with skeletal α-actin, mouse, SUMO1, mouse anti-Bcl2, Bax, cleaved caspase-3, caspase-3, cleaved PARP and PARP antibodies (all from Cell Signaling Technologies, Beverly, MA, USA), β-MHC, PPARA, NCX, anti-PLB, (Abcam, Cambridge, MA, USA), Anti-Ryanodine Receptor 2(alomone labs).