Figure 5

Mab21l2 interacts with Smad1 in vitro and in vivo. (A) Lanes 1, 2: immunoblotting of total lysates from P19 cells, mock- and Smad1-transfected, respectively. P19 cells do not express detectable levels of endogenous SMAD1. Lanes 3–8: Affinity chromatography (pull-down) experiment. High-stringency eluates from untransfected P19 were separated by SDS-PAGE and transfered onto nitrocellulose filters. The blots were immunolabeled using an anti-SMAD1 antibody. Lanes 3–6: the in vitro interaction is not strictly dependent on activation of BMP signaling: the pull-down experiment was performed with 20 μl (3, 4) and 40 μl of P19 cell lysates (5, 6). Lysates came from P19 cells treated (4, 6) or untreated (3, 5) with BMP4. Lanes 7, 8: an His-ZZ-MAB21L2 protein (HisMab) synthesized in E. coli (lane 8) was coupled to a sepharose-Ig resin and incubated with cell lysates of P19 cells treated with BMP4. As a negative control, an in-vitro synthesized His-ZZ incubated with the same P19 lysates fails to pull down a 53 kD band. Arrows: SMAD1 (53 kD). (B) direct interaction between SMAD1 and MAB21L2; no direct interaction between SMAD4 and MAB21L2. An His-ZZ-MAB21L2 protein (+Mab) synthesized in E. coli (lane 2) was coupled to a sepharose-Ig resin and incubated with in vitro-translated SMAD1 (lanes 2), SMAD4 (lanes 4), and SMAD1 + SMAD4 (lanes 6). Negative controls were represented by His-ZZ-coupled resins (-Mab) incubated with the same in vitro-synthesized proteins (lanes 3, 5, 7). Lanes 1 and 8 contain in vitro-synthesized SMAD1 and SMAD4, respectively. (C) BMP4-dependent in vivo co-immunoprecipitation of flag-SMAD1 and myc-MAB21L2 in P19 cells. Cells were mock-transfected, transfected with flag-SMAD1 and/or with myc-MAB21L2, as indicated, and either treated with BMP4 or left untreated. In lanes 3–7, cell lysates were immunoprecipitated with a monoclonal anti-flag antibody. Cell lysates in lanes 1, 2 and immunoprecipitates in lanes 3–7 were gel fractionated and transfered to nitrocellulose filters. Blots were immunolabeled with an anti myc antibody. Arrow (42 kD) points to a band in lanes 2, 3) corresponding to myc-MAB21L2. (D) in vivo co-immunoprecipitation of flag-SMAD1 and myc-MAB21L2 in stage 11 Xenopus embryos, facilitated by BMP4 overexpression. Embryos were water-injected, injected with flag-Smad1 and/or with myc-Mab21l2 RNA, as indicated, and coinjected with BMP4 where indicated. In lanes 3–7, cell lysates were immunoprecipitated with a monoclonal anti-flag antibody. Embryo lysates in lanes 1, 2 and immunoprecipitates in lanes 3–6 were gel fractionated and transfered to nitrocellulase filters. Blots were immunolabeled with an anti myc antibody. Arrow (42 kD) points to a band in lanes 2–4) corresponding to myc-MAB21L2.