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A–C
Protein extracts from MIAPaCa‐2 cells (left) and control or Tgif1
KO mice (right) were immunoprecipitated using normal IgG, anti‐TGIF1, or anti‐Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A–C). In (C), cell lysates were treated with DNase I before coimmunopreciptation. To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1, or β‐Actin.
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C
Panc‐1 cells were treated with TGF‐β for 30 min, and protein extracts were immunoprecipitated with anti‐TGIF1 antibody and analyzed by immunoblotting using anti‐Twist1 antibody. To control for activation of TGF‐β signaling, total cell lysates were analyzed by immunoblotting using anti‐pSmad2 antibody.
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D
PL45 stably expressing Dox‐inducible control or shRNA targeting TGIF1 were treated with vehicle or Dox for 48 h and analyzed for Twist1 expression by immunoblotting or qRT–PCR (n = 6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1. In right panel, data are expressed as mean ± SEM. ***P < 0.001; ns, not significant; based on a two‐tailed Student's test.
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E
PL45 stably expressing Dox‐inducible TGIF1 were treated with vehicle or Dox for 48 h and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.
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F
Pancreatic chromatin from control or Tgif1
KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.
