FIGURE

Figure 3

ID

ZDB-FIG-200523-39

Publication

Cheng et al., 2004 - Lefty blocks a subset of TGFbeta signals by antagonizing EGF-CFC coreceptors

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Figure 3

Lefty Binds to Cripto, but Not to the Activin Receptors ActRIIB and Alk4

(A and B) Lefty1 interacts with Cripto. RNAs (1 ng each) encoding ActRIIB(KR)/Myc, Alk4(KR)/Flag, Cripto/Flag, Lefty1/HA, or Sqt/HA were injected into Xenopus embryos. After chemical cross-linking, lysates were immunoprecipitated for either Lefty1/HA or Sqt/HA (A) with anti-HA antibody, or ActRIIB(KR)/Myc, Alk4(KR)/HA, Cripto/Flag (B) with, respectively, anti-Myc, anti-HA, or anti-Flag antibodies. Note that Lefty1 specifically interacts with Cripto (A and B), and these Lefty/Cripto complexes do not contain Alk4 (A). Moreover, processed Lefty1 binds much more efficiently to Cripto than full-length Lefty1 precursor (B). In contrast, Sqt can bind to ActRIIB, Alk4, and Cripto (A). The 55 kDa protein marker in (B) is estimated based on molecular weight markers.

(C) Lefty1 competes with Nodal for binding to Cripto. RNAs encoding Sqt/HA (1 ng), Cripto/Flag (100 pg), or Lefty1 (2 ng) were injected and anti-Flag antibody was used to immunopreciptate Cripto/Flag.

(D) mLefty1 binds directly to Cripto. Purified mouse Lefty1 protein (mLefty1; 10 μg/ml) was mixed with either soluble purified Cripto/His protein (5 μg/ml) or purified control VEGF-D/His protein (5 μg/ml). After chemical cross-linking, mLefty1 was immunoprecipitated with anti-mLefty1 antibody. mLefty1 associates with Cripto, but not with control VEGF-D.

Proteins in the coimmunoprecipitates and total extracts were probed in Western blot analysis with the indicated antibodies: ActRIIB(KR)/Myc (kinase-defective receptor, approximately 120 kDa; anti-Myc), Alk4(KR)/Flag (kinase-defective receptor, approximately 70 kDa; anti-Flag), Cripto/Flag (approximately 30 kDa; anti-Flag), Lefty1/HA (mature ligand, approximately 36–40 kDa; anti-HA; Sakuma et al. 2002), Sqt/HA (unprocessed precursor, approximately 55 kDa; mature ligand, approximately 22 kDa; anti-HA), Lefty1/Glu (unprocessed precursor, approximately 55 kDa; mature ligand, approximately 38 kDa; anti-Glu; Sakuma et al. 2002), mLefty1 (mature ligand, approximately 36 kDa, anti-mLefty1; Sakuma et al. 2002), Cripto/His (soluble form, approximately 22–25 kDa; anti-His), and VEGF-D/His (mature ligand, approximately 15–20 kDa; anti-His).

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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