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Figure 2


Fig. 2. Antimycin A and the adipogenic cocktail trigger CREB activation by phosphorylation on Ser133. (A) Western blot analysis for CREB and CREB phosphorylated on Ser133 (pCREB) performed with 20 µg of nuclear proteins extracted from 3T3-L1 cells incubated for 6, 24 and 48 hours, and 3 and 8 days without a drug (controls, CTL), with 10 nM AA or with the adipogenic cocktail (DIFF); TBP served as a loading control. (B) Immunostaining and confocal microscopic analysis of abundance and localization of pCREB in 3T3-L1 cells incubated for 24 hours (a) without, (b) with 10 nM AA or (c) with adipogenic cocktail. Arrows indicate nuclear localization of pCREB. (C) Quantitative detection of pCREB and total CREB that binds to a DNA consensus sequence in a colorimetric assay (TransAM kit). Assays were performed on 10 µg of nuclear protein extract from 3T3-L1 cells incubated for 24 hours without (CTL), with 10 nM AA or with adipogenic cocktail (DIFF). Phosphorylated and total forms of CREB binding of pCREB and total CREB to DNA was detected with anti-Ser133-pCREB or anti-CREB antibodies, respectively, followed by a colorimetric reaction in the presence of a HRP-conjugated secondary antibody. Absorbance values were measured at 450 nm for DNA-binding of pCREB and CREB, and signals obtained for pCREB were normalized against total CREB bound to the capture probe (n=3). (D) Effect of AA and the adipogenic cocktail on CREB transcriptional activity in 3T3-L1. Cells were transiently co-transfected with a plasmid encoding ß-galactosidase and a CREB-sensitive luciferase-reporter construct. The next day, cells were incubated for 24 hours without (CTL) or with 10 nM AA, or with the adipogenic cocktail (DIFF) and then processed for luciferase assay. Results were normalized against ß-galactosidase activity and expressed as fold-increase of controls as the mean ± s.d. for n=3 experiments. *P<0.05, **P<0.01, ***P<0.001 are significantly different from control cells.





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