Immunoblots for nonphosphoproteins were carried out on the same membranes used to evaluate the phosphorylated (
phospho-) forms: phospho-MYPT-1 ([Thr.sup.853]), phospho-CPI-17 ([Thr.sup.38]), phospho-MLC ([Thr.sup.18]/[Ser.sup.19]), and phospho-AMPK ([Thr.sup.172]), (1:500; Cell Signaling, USA).
Louis, MI, USA) followed by overnight incubation at 4[degrees]C with the following primary antibodies: SIRT1, SIRT2, and SIRT3 (1 : 1000, Cell Signalling Technology, Beverly, MA, USA); I[kappa]B-[beta] (dilution 1:500, Delta Biolabs Muraoka Drive Gilroy, CA, USA); InB-a and
phospho- I[kappa]B-[alpha] (both 1:1000, Cell Signalling Technology, Beverly, MA, USA); PBEF (1:5000, Bethyl Laboratories Inc.