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Fig. 5 | Cell Communication and Signaling

Fig. 5

From: Sirt1 interaction with active Smad2 modulates transforming growth factor-β regulated transcription

Fig. 5

Sirt1 detection on different regulatory elements is increased after TGFβ treatment. Nuclear extracts from HaCaT cells treated or not with TGFβ for 1 h were used for DNA pull-down assays. a Extracts were supplemented with Sirt1-6xHis and HA-Mixer-transfected HEK293T nuclear extracts. Pull-down was performed with specific oligonucleotides for regulatory sequence DE-goosecoid and mutant oligonucleotides for Smad2/Smad4 binding. Antibodies used for Western Blot detection are indicated. Pull down Sirt1-6xHis (PD His) and total Sirt1-6xHis were quantified. PD His/T His is indicated (lane 9–12 over lane 9). b Extracts were supplemented with Sirt1-6xHis. Pull-down was performed with specific oligonucleotides for regulatory sequence c-jun-SBR and mutant oligonucleotides for Smad3/Smad4 binding. The indicated antibodies were used for Western Blot detection. Pull down Sirt1-6xHis (PD His) and total Sirt1-6xHis were quantified. PD His/T His is indicated (lane 5–10 over lane 5). All the experiments of this figure were repeated at least three times. Representative results are shown. WT: Wild Type oligonucleotide. Mut: mutant oligonucleotide. Buffer: no-input control. DNAP: DNA pull-down. NT: not transfected. (*): unspecific band

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