Lactate dehydrogenase

Soluble protein lysates were prepared as previously described (20 (link)). Thirty micrograms of protein per independent sample was resolved using SDS-polyacrylamide gels (10 or 12%) and transferred to PVDF membranes using an electroblotting method. The membranes were incubated with blocking buffer (5% dried milk in Tris buffered saline with Tween-20 (TBST)) at room temperature, washed with TBST and then incubated with primary antibodies against either, human PPARβ/δ, cellular retinoic acid binding protein II (CRABP-II; Abcam, Cambridge, MA), p65, proliferating cellular nuclear antigen (PCNA), β-ACTIN (Santa Cruz Biotechnologies, Santa Cruz, CA), fatty acid binding protein 5 (FABP5; Biovendor, Chandler, NC), poly (ADP-ribose) polymerase (PARP; Cell Signaling Technology, Danvers, MA), or lactate dehydrogenase (LDH, Rockland, Gilbertsville, PA) at 4°C overnight. After washing three times with TBST at room temperature for 10 minutes each, the membranes were incubated with biotin-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature and then with ¹²⁵I-streptavidin. The membranes were exposed to phosporimager plates and the level of radioactivity quantified with filmless autoradiographic analysis (Packard Phosphorimager, PerkinElmer, Waltham, MA). Hybridization signals for specific proteins were normalized to that of LDH or β-ACTIN.