Pgc 1α nbp1 04676

Total liver tissue lysates were fractioned by 10% acrylamide gel. The proteins were then transferred to a nitrocellulose membrane and blocked in 5% milk protein diluted in TBST (0.1%) for 1 h. All primary Ab incubations were overnight: NF-κB p65 (8242s; Cell Signaling Technology), acetyl–NF-κB p65 (Lys³¹⁰) (3045s; Cell Signaling Technology), β-actin (A5441; Sigma-Aldrich), acetyllysine (ab80178; Abcam), IKBα (4814S; Cell Signaling Technology), Sirt1 (07-131; MilliporeSigma), PPAR γ coactivator-1a1 (PGC-1α; NBP1-04676; Novus Biologicals), and PBEF (nicotinamide phosphoribosyltransferase [NAMPT]) (sc-393444; Santa Cruz Biotechnology). Next day, membranes were washed in TBST and incubated with secondary Abs (anti-mouse (A4416; Sigma-Aldrich) and anti-rabbit (7074S; Cell Signaling Technology) for 1 h. Proteins were detected with chemiluminescence (Amersham Bioscience).

Immunoblot and immunofluorescence assays were performed following standard procedures, as previously described [40 (link)]. Primary antibodies were: phospho Histone H3 Ser10 (ab14955, Abcam), TOMM20 (11802-1-AP, Proteintech), PGC1-α (NBP1-04676, Novus Biologicals), phospho p38MAPK Thr180/Tyr182 (9211, Cell Signaling), p38MAPK (sc-7972, Santa Cruz Biotechnology) AKT1/2/3 (sc-8312, Santa Cruz Biotechnology), phospho AKT Ser 473 (9271, Cell Signaling), DMPK (sc-134319, Santa Cruz Biotechnology), MBNL1 (ab45899, Abcam) and β-actin (AC-15, Sigma-Aldrich). For western blot detection of primary antibodies, we used HRP-linked antibodies (Santa Cruz Biotechnology) and the detection was performed by chemiluminescence using Novex ECL Chemi Substrate (Thermo Fisher). For immunofluorescence, nuclear DNA was stained with Hoechst (33342, Sigma-Aldrich).