At 48 h after transfecting 6 105 COS1 cells, cells were incubated for 30 min on ice in 200 l TNE buffer (25 mM TrisCHCl pH 7.4, 150 mM NaCl, 5 mM EDTA) containing protease inhibitors (Complete Mini, Roche) and 1% Triton X-100. the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to Proc extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFPCFlotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells. synthesis was blocked by cycloheximide (CHX, 5 g/ml). (B) BFA inhibited the secretion of Nodal into the medium, leading to sequestration of the 47-kDa form in cell lysates. Cripto independently interacts with processed Nodal and its pro peptide To test whether Nodal binds Cripto before proteolytic maturation, a mixture of soluble Nodal propeptide, mature form and residual uncleaved precursor (Figure 4A, top panels) was incubated with metal agarose beads soaked with or without CriptoHis. CriptoHis mainly pulled down Telotristat uncleaved Nodal (top panels), despite a molar excess of processed Nodal and propeptide in the input (bottom panels). However, when incubated Telotristat with Nodal-sc, which is more efficiently processed due to a mutant cleavage site (Constam and Robertson, 1999), CriptoHis pulled down both mature Nodal and propeptide (lane 2). These results indicate that Nodal binds Cripto before and after proteolytic maturation. Open in a separate window Figure 4 Cripto binds mature and uncleaved Nodal, and a Cripto-interacting region in the propeptide ensures that Nodal processing enhances signalling. (A) Pull down of Flag-tagged Nodal precursor and cleaved fragments (pro, mat) by CriptoHis. Supernatant of 293T cell lines containing Nodal propeptide, mature form and residual uncleaved precursor (bottom panels, input) was incubated with metal agarose beads soaked with (lanes 1 and 2) or without (lanes 3 and 4) CriptoHis. Western blot analysis revealed that CriptoHis (18C28 kDa) precipitates Nodal precursor (pre), together with small amounts of propeptide (pro) and mature form (mat, lane 1). Mutation of the PC recognition motif RQRRHHL to RQRRHLE in supercleaved Nodal-sc (Constam and Robertson, 1999) increased binding of Cripto to mature Nodal and propeptide (lane 2). Mature Nodal (12 kDa) and precursor (47 kDa) were shifted in size by 6 kDa due to an N-glycosylation site engineered to improve protein stability (Le Good mutant embryos when injected at non-physiologically high concentrations (Minchiotti hybridization Embryos were dissected 6.5 days post-coitum and fixed overnight at 4C in PBS containing 4% paraformaldehyde and 0.1% v/v Tween-20. DIG-labelled Nodal and fluorescein-labelled Cripto antisense mRNA probes were hybridized to whole mount embryos and sequentially revealed using BM purple and INT/BCIP to detect alkaline phosphatase conjugated to anti-DIG and anti-FLUO antibodies (Roche), respectively. Frozen sections (8 m) were obtained after embedding stained embryos in glycerol containing 30% (w/v) sucrose. Density fractionation gradients All of the following steps were performed in the cold room. At 48 h after transfecting 6 105 COS1 cells, cells were incubated for 30 min on ice in 200 l TNE buffer (25 mM TrisCHCl Telotristat pH 7.4, 150 mM NaCl, 5 mM EDTA) containing protease inhibitors (Complete Mini, Roche) and 1% Triton X-100. The resulting cell suspensions were passed 10 times through a 26G needle, and protein concentrations were adjusted to 1 1.2 g/ml. Of the resulting homogenates, 90 l was thoroughly mixed with 120 l of 60% Optiprep (stock solution; Axon Lab) and then overlaid without mixing by 2 ml of 30% Optiprep (diluted in TNE) and 190 l of TNE buffer. After centrifugation for 2 h (55 000 r.p.m., 25 9000 at 4C), six 400 l fractions were precipitated by adding sodium deoxycholate Telotristat (125 g/ml) and trichloroacetic acid (4.8 mg/l). Telotristat After resuspension in SDS sample buffer, proteins of interest were visualized in each fraction by western blot analysis. Supplementary Material Supplementary Figure S1 Click here to view.(1.4M, tiff) Supplementary Figure S2 Click here to view.(887K, tiff) Supplementary Figure S3 Click here to view.(1.7M, tiff) Supplementary data Click here to view.(56K,.
At 48 h after transfecting 6 105 COS1 cells, cells were incubated for 30 min on ice in 200 l TNE buffer (25 mM TrisCHCl pH 7