Figure 4.

TGF-β1 induces ALK1 hetero-oligomerization with ALK5 and subsequent phosphorylation of Smad1. DIV14 hippocampal neurons (A) or secondary astrocytes (B) were incubated with the indicated cytokines, lysed, and subjected to SDS-PAGE and blotting (as described in Fig. 3 A). Membranes were incubated with P-Smad1 antibody. An unspecific band served as internal loading control as previously described (Goumans et al., 2002; compare B to Fig. 3 E, stripped membrane). The experiment was performed in triplicate with comparable results (A). (C) Immunofluorescence detection of Smad1 translocation to the nucleus and its quantitative evaluation in cultured rat hippocampal neurons. Neurons were treated with 10 ng/ml TGF-β1 for 3 h. After fixation and permeabilization, localization of Smad1 protein was determined by anti-Smad1 antibody (red). Neuronal nuclei were stained using NeuN antibody (green). Note the increase in Smad1 nuclear patches occurring in TGF-β1–treated cells, resembling neurons treated with BMP-4 (Angley et al., 2003). Bars, 10 μm. Quantification of nuclear translocation was determined without knowledge of the respective treatments. A total of 350–450 cells were counted in three separate experiments. Data are means ± SEM; *, P < 0.05. (D) Coimmunoprecipitation analysis of TGF-β1 induced ALK5/ALK1 receptor complexes. Cortical neurons (DIV14) were treated with10 ng/ml TGF-β1 for 15 min. After immunoprecipitation with ALK5 antibody, SDS-PAGE and Western blotting were conducted as described in Fig. 3 A. Membranes were incubated with the anti-ALK1 antibody. Immunodetection of light-chain (LC) by anti–rabbit antibody served as control. (E) Hippocampal neurons were incubated with 10 ng/ml TGF-β1 for 1 h. 30 min of preincubation with 5 μM SB-431542 were performed as indicated.