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. 2002 Sep 16;158(6):1067–1078. doi: 10.1083/jcb.200204063

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Copyright © 2002, The Rockefeller University Press

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Figure 6. — Analysis of complex formation, subcellular localization, and effects on transcriptional activity using combinations of APC-25, Axin, and GSK3β with β-cat/TCF4. (A) Expression vectors for APC-25, Axin, and GSK3β were introduced into yeast cells containing stably integrated β-cat and TCF4. β-cat was immunoprecipitated from protein extracts followed by Western blot with antisera against the HA tag (to detect APC-25 and Axin), human GSK3β, the FLAG tag (to detect TCF4), or β-cat in total extracts (T) and β-cat immunoprecipitates (IP). (B and C) Cells containing integrated β-cat and TCF4 alone (A–C and M–O) or with Axin (D–F and P–R), GSK3β (G–I and S–U), or Axin plus GSK3β (J–L and V–X) and with either a control vector (Control) or an APC-25 expression vector (APC-25) were subjected to IF with an antibody against β-cat (B) or the HA tag (to detect Axin or APC-25) (C). Proteins (columns 1and 4), DNA (columns 2 and 5), and cells (columns 3 and 6) were photographed. (D) Yeast cells with integrated β-cat and TCF4 and combinations of APC-25, Axin, and GSK3β as well as the 5X LEF LacZ reporter were assayed for β-gal activity.

Figure 6. — Analysis of complex formation, subcellular localization, and effects on transcriptional activity using combinations of APC-25, Axin, and GSK3β with β-cat/TCF4. (A) Expression vectors for APC-25, Axin, and GSK3β were introduced into yeast cells containing stably integrated β-cat and TCF4. β-cat was immunoprecipitated from protein extracts followed by Western blot with antisera against the HA tag (to detect APC-25 and Axin), human GSK3β, the FLAG tag (to detect TCF4), or β-cat in total extracts (T) and β-cat immunoprecipitates (IP). (B and C) Cells containing integrated β-cat and TCF4 alone (A–C and M–O) or with Axin (D–F and P–R), GSK3β (G–I and S–U), or Axin plus GSK3β (J–L and V–X) and with either a control vector (Control) or an APC-25 expression vector (APC-25) were subjected to IF with an antibody against β-cat (B) or the HA tag (to detect Axin or APC-25) (C). Proteins (columns 1and 4), DNA (columns 2 and 5), and cells (columns 3 and 6) were photographed. (D) Yeast cells with integrated β-cat and TCF4 and combinations of APC-25, Axin, and GSK3β as well as the 5X LEF LacZ reporter were assayed for β-gal activity.