Ecl immunoblotting detection system

Protein expression analysis was obtained from four mice from the control group and 4 mice from the ghrelin-treated group. Frozen hypothalamus and cortex were homogenized in protein extraction buffer (30 mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% sodium deoxycholate [DOC], 1% Triton X-100 with phosphatase and protease inhibitors). Fifty micrograms of protein were analyzed on 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: GAD65/GAD2 (1/1000; Cell Signaling ref. 5843), VGLUT2 (1/1000; Cell Signaling ref. 71555), and β-actin (1/50,000; Sigma-Aldrich). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an ImageQuant LAS4000 mini Fuji luminescence imagining system. The bands were quantified by densitometry using ImageJ analysis software.

Cells were harvested in lysis buffer containing: 10 mM Tris pH 8, 10 mM MgCl₂, 0.2 mg/ml AEBSF (MegaPharm-101500), 0.5 mg/ml Lysozyme (USBiological-L9200), 5 µg/ml DnaseI (Sigma DN25). Protein concentrations were determined using Bradford analyses. Samples were separated on 10% SDS-PAGE gels, and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: Polyclonal anti-yeast fumarase and anti-human FH were generated in rabbits injected with the purified proteins. Monoclonal anti GFP was a product of Roche). Monoclonal anti SigmaA was kindly provided by M. Fujitas lab,. Polyclonal anti ICDH and anti citZ were product of kerafast. Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an ImageQuant LAS4000 mini Fuji luminescence imagining system. For the analysis of protein expression, bands from at least three independent experiments were quantified by densitometry using Image J analysis software.

rBA were cultured in 12-well plates, differentiated and infected as described above. Cells were harvested in lysis buffer (RIPA), and protein concentration was determined using a BCA protein assay kit (Thermoscientific). Samples were separated on 8% and 12% SDS-PAGE gels, for CPT1A and UCP1 respectively, and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: CPT1A (1/6,000) [24 (link)], Tim 44 (1/5,000; BD Bioscience), UCP1 (1/1,000; Abcam) and b-actin (I-19; 1/4,000; Santa Cruz). Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an ImageQuant LAS4000 mini Fuji luminescence imagining system. For the analysis of protein expression, bands from at least three independent experiments were quantified by densitometry using Image J analysis software.