FIGURE 5.

Determination of the TGF-β receptor-interacting domain in TMED10. A, illustration of TMED10 and its mutants. SP, signal peptide; TM, transmembrane. B, interaction of TMED10 or its mutants with ALK5. COS7 cells were transfected with the indicated plasmids and harvested for co-IP experiments. The interaction between ALK5 and either TMED10 or its mutants is shown in the top panel. Total expressions of ALK5ca/V5 and TMED10/FLAG (or its mutants) are shown in the middle and bottom panel, respectively. C, non-necessity of both the transmembrane and the intracellular domains of TMED10 to interact with ALK5. The COS7 cells were transfected with the indicated plasmids. Section I, after cell lysis, anti-V5 antibodies were used for immunoprecipitation. The immunoprecipitates were separated by SDS-PAGE followed by Western blotting analysis (WB) using anti-FLAG antibody to detect their interaction (top panel). Total expressions of ALK5ca/FLAG and TMED10/V5 (or its mutants) in cells are shown in the second and third panel, respectively. Section II, because TMED10Δ(V¹⁸⁶-E²¹⁹) seemed to be a secretory protein, immunoprecipitation of TMED10Δ(V¹⁸⁶-E²¹⁹) into the media in which the cells were cultured was performed to detect secretory TMED10Δ(V¹⁸⁶-E²¹⁹) (bottom panel). D, interaction of TMED10 or its mutants with TβRII. Co-IP was performed as described in B above. The interaction between TβRII and TMED10 or it mutants is shown in the top panel. Total expressions of TβRII and TMED10 or its mutants were detected using anti-HA12CA5 (middle panel) and anti-FLAG antibodies (bottom panel), respectively. E, schematic presentation of GST-TMED10 fusion proteins. The region from Gly⁸¹ to Val¹³⁰ in TMED10 was split into two pieces to make GST fusion proteins. F, requirement of the region consisting of 30 amino acids from Gly⁸¹ to Glu¹¹⁰ within TMED10 to interact with ALK5. A GST pulldown assay was performed using the GST fusion proteins described in E above. ALK5 ectopically expressed in COS7 cells was mixed with GST fusion protein. After loading the protein(s) bound to GSH-Sepharose 4B in SDS-PAGE, Western blotting analysis was performed using anti-ALK5 (V-22) antibody (top panel). The cell lysates from COS7 cells transfected with pcDNA3 were used as the negative control. As loading controls, GST and GST fusion proteins were visible with Ponceau S staining (bottom panel). G, illustration of GST-TMED10 fusion proteins. The region from Gly⁸¹ to Glu¹¹⁰ in TMED10 was divided into two pieces to make GST fusion proteins. H, determination of the ALK5-binding region in TMED10. A GST pulldown assay was carried out as described in F above. The top and bottom panels show the interaction of ALK5 with GST fusion proteins and the loading controls with Ponceau S staining. I, requirement of a 20-amino acid-long region in TMED10 to associate with TβRII. A GST pulldown assay was carried out as described in F above. The upper and lower panels show the interaction of TβRII with GST fusion proteins and the loading controls with Ponceau S staining.