Myocardial tissue and cardiomyocytes were lysed with RIPA buffer for the determination of protein arginine N-methyltransferase 1 (PRMT1), dimethylarginine dimethylaminohydrolase1 (DDAH1) or DDAH2, eNOS, PGC-1α, UCP2 and β-actin (the former three antibodies were products of Abcam, Cambridge, MA, USA from rabbit, goat, goat, respectively; and the latter four antibodies from Santa Cruz Biotechnology, Santa Cruz, CA, USA from rabbit, rabbit, goat, rabbit, respectively) expression by Western blotting as described previously [25 (link)].
Immunoprecipitation was employed to detect post-translational acetylation and phosphorylation of PGC-1α as described by Lei S et al [28 (link)]. In this experiment, a total of 400 μl cell lysates were subjected to immunoprecipitation with 1 μg PGC-1α primary antibody at 4 °C overnight. The antibody-bound proteins were precipitated with 15 μl protein G magnetic beads. After separating from protein G magnetic beads, immunoprecipitates were subjected to Western blotting as described above with the antibody against acetylated-lysine or phosphorylated-serine/threonine (protein G magnetic beads and antibodies were products of Cell signaling technology, Danvers, MA, USA, the latter from rabbit), respectively.
Immunoprecipitation was employed to detect post-translational acetylation and phosphorylation of PGC-1α as described by Lei S et al [28 (link)]. In this experiment, a total of 400 μl cell lysates were subjected to immunoprecipitation with 1 μg PGC-1α primary antibody at 4 °C overnight. The antibody-bound proteins were precipitated with 15 μl protein G magnetic beads. After separating from protein G magnetic beads, immunoprecipitates were subjected to Western blotting as described above with the antibody against acetylated-lysine or phosphorylated-serine/threonine (protein G magnetic beads and antibodies were products of Cell signaling technology, Danvers, MA, USA, the latter from rabbit), respectively.
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