β actin

Ovarian tissue protein extracts (30 μg) were separated by SDS-PAGE using 13 % (v/v) gels and transferred onto a PVDF membrane. Membranes were blocked with 5 % (v/v) non-fat dry milk overnight at 4 °C and then washed for 10 min with the washing buffer (0.1 % v/v Tween 20, 50 mM Tris/HCl, and 200 mM NaCl; pH 7.6). The membranes were incubated for 2 h with primary antibodies (dilution 1:1,000 in blocking buffer) that specifically recognised the active form of TIMP-2 and (Santa Cruz Biotechnology Inc., Texas, USA), TIMP-3, (Santa Cruz), and β-actin (Santa Cruz). After primary antibody binding, the membranes were washed three times for 15 min each with TBS-T buffer and incubated for 2 h with HRP-conjugated anti-goat (Abcam, Cambridge, UK) or anti-mouse (Abcam) secondary antibodies diluted 1:5,000 in blocking buffer). The membranes were incubated for 5 min with the ECL detection reagent in the dark and then exposed to X-ray film for 10 min; protein expression was normalized to that of β-actin, which served as an internal control, using the software Alpha Innotech ver. 4.0.0 (San Leandro, CA, USA).

Proteins from the cytosolic fraction (65 µg) were separated on 4–20% SDS-polyacrylamide gels (PAGE) (Bio-Rad), and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad). Cytosolic protein blots were incubated overnight in Tris-buffered-saline containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies for proinflammatory cytokines: IL-1β (1∶500), GFAP (1∶1000), cPLA₂-IVA, sPLA₂-IIA, iPLA₂-VIA, COX-1 (1∶500) and COX-2 (1∶200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and β-actin (1∶10,000) as a housekeeping protein (Sigma Aldrich). The cytosolic blots were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad), and were visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ, USA). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA, USA) and were normalized to β-actin. All experiments were conducted on six independent samples. Mean values are expressed as percent of control.

Cellular total protein and mitochondrial and cytosolic protein fractions, were isolated as previously described [26] (link), [32] (link). Protein concentrations were determined by the Bio-Rad (Hercules, CA) protein assay according to the manufacturer’s instructions. Western blotting was performed, as previously described [33] (link), except transfers were performed with a Bio-Rad Trans-Blot Turbo Transfer System. Nitrocellulose membranes were exposed to antibodies that recognize phospho-Ser-16-stathmin, phospho-Ser-38-stathmin, total JNK, phosphorylated c-Jun, phosphorylated and total ERK1/2, phosphorylated MAPK kinase 4, caspase 3 and 7, tubulin (Cell Signaling, Beverly, MA), phosphorylated JNK, total c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), total stathmin (EMD Millipore, Billerica, MA), phospho-Ser-25-stathmin (Abcam, Cambridge, MA), β-actin (Sigma), cytochrome c (BD Biosciences) and cytochrome oxidase (MitoSciences, Eugene, OR). Western blot signals were quantitated by a FluorChem densitometer (Alpha Innotech), and stathmin signals were normalized to that for β-actin.

Cells were grown to 60-80% confluence in 60 mm round petri dishes. Following drug treatment, cell lysates were collected for protein quantification using the Bradford method (16 (link)). For tissues, brains were dissected from healthy Sprague Dawley rats following administration with R481 (20 mg kg^-1; i.p), R419 (20 mg kg^-1; i.p) or vehicle (0.5% HPMC, 0.1% TWEEN-80; i.p) during glucose tolerance tests (2 g kg^-1) 120 minutes following drug administration and snap frozen in liquid nitrogen. 2 mm sections encompassing the medial basal hypothalamus (MBH) were cut using the rat brain matrix (A1TO 1mm World Precision instruments) and MBH dissected with a scalpel. Protein was extracted from the region of interest by mechanical homogenisation, in lysis buffer, and quantified using the Bradford method. Extracted protein was separated using SDS-PAGE and transferred to nitrocellulose membranes. Immunoreactivity for total and phosphorylated protein was detected and semi-quantified using infrared fluorescence on the Licor Odyssey scanner. Primary antibodies used were: pThr172 AMPK (1:1,000; catalogue #2531), AMPKα (total AMPK 1:1000, catalogue #2535), pSer79 acetyl CoA carboxylase (ACC; 1:1000; catalogue #3661) from Cell Signalling Technologies, total ACC (1:1,000; catalogue #05-1098) from Merck Millipore and β-actin (1:10,000; catalogue #NB600-501) from Biotechne.