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. 2005 Oct;25(20):8864–8873. doi: 10.1128/MCB.25.20.8864-8873.2005

FIG. 2.

FIG. 2.

Protein-protein interaction between GATA6 and LMCD1/dyxin. (A) GST pull-down assay showing the interaction between GATA6 and full-length LMCD1/dyxin. (B) Coimmunoprecipitation of GATA6 and LMCD1 from MLE-15 cell extracts. Extracts were immunoprecipitated with a GATA6 antibody and then analyzed by Western blotting with the LMCD1/dyxin polyclonal antibody. “Input” represents 5% of the extract used in the immunoprecipitation. Position of the 35-kDa molecular mass marker is shown. Expression of GATA6 in MLE-15 cells has already been reported (6, 31). IP, immunoprecipitation; WB, Western blot; NIgG, normal IgG; G6 Ab, GATA6 antibody. (C) GST pull-down assay showing interaction between the LIM and CYS domains and GATA6. Arrowheads in panels A and C indicate radiolabeled GATA6 interaction with GST-LMCD1 fusion proteins. (D) Coomassie blue-stained gel of GST fusion proteins. Asterisks denote full-length fusion proteins. Molecular mass standards in kilodaltons are noted on the left. (E) Far-Western blot showing interaction between GATA6 and the LIM- and cysteine-rich domains of LMCD1/dyxin. The arrow and asterisks denote GATA6 interaction with GST fusion proteins. The arrowhead denotes a nonspecific band that reacts with anti-GST-LMCD1 antisera. Mock-transfected cell extracts were probed with full-length GST-LMCD1. Molecular mass standards in kilodaltons are noted on the left. (F) Summary of the domains within LMCD1/dyxin that interact with GATA6. −, no interaction; +++, interaction. (G) GST pull-down assay to assess the abilities of different regions of GATA6 that interact with LMCD1/dyxin, as well as full-length GATA1 and GATA4. Molecular mass standards in kilodaltons are noted on the left. (H) Input of radiolabeled GATA6, GATA1, and GATA4 proteins. Molecular mass standards in kilodaltons are noted on the left. (I) Summary of domains within GATA6 that interact with LMCD1/dyxin. These regions delineate the carboxy-terminal zinc finger as the domain in GATA6 required for interaction with LMCD1/dyxin. Gel lane numbers in panels G and H correspond to the GATA6 proteins indicated in panel I.