JCS03074 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. RGMc is a disulfide-linked protein. (A) Detection of adenoviral-derived HA-RGMc by immunoblotting (arrows) after crosslinking of cell-surface proteins by EZ-link and capture by ‘pull-down’ with streptavidin agarose (EZ), or after release from the cell surface by incubation with PI-PLC. Results are shown after reducing (left panel) and non-reducing (right panel) SDS-PAGE. (B) Immunoblotting with whole-cell protein extracts (WCE) and conditioned culture media (CM) from Cos-7 cells infected with Ad-HA-RGMc (wt) or Ad-HA-RGMcΔGPI (Δ) and separated under reducing (left panel) or non-reducing (right panel) SDS-PAGE.

• Supplemental Figure 2 -

  Fig. S2. Detection of cell-associated and secreted RGMc using different reducing and denaturing conditions. Results are shown of immunoblotting with whole cell protein extracts (WCE) and conditioned culture media (CM) from Cos-7 cells infected with Ad-HA-RGMc. Before SDS-PAGE protein samples were incubated with different combinations of reducing agents (100 mM DTT, 1% β-mercaptoethanol), denaturant (8 M urea), and cysteine alkylating reagent iodoacetamide (IAN, 10 mM). Note that all treatments led to identical results.

• Supplemental Figure 3 -

  Fig. S3. Secreted RGMc is stable following incubation at low pH. Conditioned media (10 μl) from undifferentiated C2 myoblasts infected with Ad-HA-RGMc was incubated for 2 hours at 37°C in 100 mM phosphate buffer (pH 5.0 to 8.0) or 100 mM glycine pH 2.5, followed by immunoblotting with an antibody to RGMc.