Sequential actions of ERK1/2 on the AP-1 transcription factor allow temporal integration of metabolic signals in pancreatic ß cells FASEB J. Glauser and Schlegel
21: 3240
has been used to quantify the in vitro FOXO sequence-specific DNA-binding activity in nuclear extracts. A) Validation of the assay. Nuclear extracts (5 μg) from MIN6 cells were analyzed in parallel with nuclear extracts (5 μg) from raji cells (positive control) and a no template control condition (NTC). Sequence specificity of the binding was assessed by addition of soluble competitor oligonucleotides. FOXO activity in MIN6 cell nucleus is detectable (far above the NTC background signal), but low in comparison to the raji cells. The signal is specific to the DNA sequence as it was strongly decreased by the wild-type soluble oligo (WT), but not by the mutant oligo. ND, not determined. B) Nuclear extracts from MIN6 cells were prepared after 24 hours of culture at indicated concentrations of glucose and FCS. The difference of treatment does not produce a differential activation of FOXO.
Supplemental Figure 2
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(06-7798SupplementalFig2.tif; 533 KB)
p38 MAPK activation was assessed by western analysis with p38 and phopho-p38 antibodies.
A) Whole cell lysates from MIN6 cells were prepared after 24 hours of culture at indicated concentrations of glucose and FCS. B) Densitometric analysis. Mean of A) with SD as error bars, n=3. The results show no difference in p38 activation. C) The stress situation caused by an hyperosmotic shock was used as positive control. After 24 hours of culture at 10 mM glucose and 15% FCS, the hyperosmotic shock was made by adding 200 mM NaCl and incubating 40 minutes before preparing the cell lysates.
Supplemental Figure 3
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(06-7798SupplementalFig3.tif; 114 KB)
MIN6 cells were co-stimulated with 10 mM glucose and 10 nM GLP 1. Pretreatment with act D or vehicle (DMSO) was made 1h before stimulation. c-fos mRNA was quantified by real-time RT-PCR and expressed as mean of triplicates with SD as error bars. The pretreatment with actinomycin D blocks completely the transcriptional induction of c-fos.
