Anti pkac

Isoproterenol and Trichostatin A (TSA) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used at 10 µM and 100 nM, respectively. Murine TNF-α was a gift from the VIB Department for Molecular Biomedical Research of Ghent University (VIB-UGent, Gent, Belgium) and was used at 2000 IU/ml. Insulin-like growth factor-1 (IGF-1) was from ImmunoTools (Friesoythe, Germany) and was used at 10 ng/ml. Anti-β₂-AR, anti-TNF-R1, anti-myogenin, anti-PARP, anti-P-H3-Ser10, anti-CBP, anti-RNA polymerase II, anti-p65, anti-IκBα, anti-P-CREB-Ser133 and anti-PKAc were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-P-p65-Ser536, anti-P-ERK-Thr202/Tyr204, anti-P-JNK-Thr183/Tyr185, anti-P-p38-Thr180/Tyr182, anti-P-MSK-1-Thr581, anti-Lamin A/C and anti-CREB were from Cell Signaling Technology (Danvers, MA, USA). Anti-Ac-H3-Lys27 and GAPDH were from AbCam (Cambridge, UK). Anti-α-tubulin and anti-α-actin were from Sigma-Aldrich. In figures, the expression “antibody anti-” was substituted by the Greek letter “α-”. AatII and HincII restriction enzymes were obtained from New England BioLabs (Ipswich, MA, USA).

Proteins were extracted following the procedure described previously (22 (link)). The proteins were separated by SDS-PAGE on 8–12% polyacrylamide gels and subsequently electrically transferred to a PVDF membrane. After blocking with 5% (w/v) BSA in TBST at room temperature for 1 h, the membranes were then incubated with an appropriate specific primary antibody (anti-CYP7A1, Santa Cruz, Cat# sc-518007, 1:500; anti-PER1, Sigma-Aldrich, Cat# AB2201, 1:200; anti-CYP8B1, Santa Cruz, Cat# sc-101387,1:500; anti-CYP27A1, Santa Cruz, Cat# sc-390974,1:500; anti-HNF-4α, Santa Cruz, Cat# sc-101059,1:500; anti-PKA-C, Santa Cruz, Cat# sc-365615,1:500; anti-PKA-R, Santa Cruz, Cat# sc-271125,1:500; anti-β-actin, BioWorld, Cat# AP0060, 1:1000; anti-p-Ser, Santa Cruz, Cat# sc-81514) at 4°C overnight, followed by incubation with an HRP-conjugated secondary antibody (1:4000). Detection was performed using an enhanced chemiluminescence kit (Thermo Scientific, Hudson, NH).