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. 2004 Mar;24(6):2251–2262. doi: 10.1128/MCB.24.6.2251-2262.2004

FIG. 2.

FIG. 2.

Mapping of Smad7 domains which interact with Jab1/CSN5. (A) The PY motif of Smad7 is not required for Smad7-Jab1/CSN5 interaction. 293T cells were transfected with GST or GST-Jab1/CSN5 either alone or together with wild-type or ΔPY versions of Smad7. Cell extracts were subjected to GST pull-down assay with glutathione-Sepharose beads followed by immunoblotting with anti-HA antibody. Expression of GST, GST-Jab1/CSN5, HA-Smad7, and HA-Smad7ΔPY was monitored as indicated. (B) Jab1/CSN5 associates with the C-terminal domain of Smad7. FLAG-tagged Smad7 deletion mutants (7C; amino acids 204 to 407 and the N-terminal domain of Smad7; amino acids 1 to 261) (25) were cotransfected into the 293T cells together with either GST or GST-Jab1/CSN5. Cell extracts were subjected to GST pull-down assay with glutathione-Sepharose beads followed by immunoblotting with anti-FLAG antibody. Expression of Smad7 deletion mutants, GST, and GST-Jab1 was confirmed by immunoblotting aliquots of total cell lysates (bottom panels). The higher band in lanes 2 and 4 is a nonspecific band.