JCS019745 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Localization of Furin and Mint3 in HeLa cells. Cells transfected with Furin were fixed and immunostained with antibodies to Furin and Mint3 as indicated. Scale bar: 5 µm.

• Supplemental Figure S2 -

  Fig. S2. Knockdown of Mint3 does not affect the localization of the Golgi marker GM130. HeLa cells transiently expressing Furin or Mint3 RNAi 1 were immunostained with anti-Furin and anti-GM130 (Abcam), and viewed by confocal microscopy. Scale bars: 5 µm

• Supplemental Figure S3 -

  Fig. S3. The enzyme activity of Furin at the cell surface was assayed using control and RNAi-treated 293T cells. After 48 hours of transient transfection, cells were suspended and washed three times with cold PBS. Activity of Furin at the cell surface was then assayed using the fluorogenic substrate pyrArg-Thr-Lys-Arg-7-amino-4-methylcoumarin (MCA) (Liu et al., 2004). Asterisks denote significant differences from control cells (P<0.05), as determined with a Student’s t-test.

• Supplemental Figure S4 -

  Fig. S4. The acidic cluster and the hydrophobic motif of Furin are required for its direct interaction with Mint3. The GST-fusion mutants with acidic peptide deletion (Δ769-780) fail to bind recombinant His-Mint3 efficiently, and the mutants with LI/AN Y/A substitutions decrease the binding affinity between Furin and His-Mint3. His-tagged Mint3 fusion proteins were immobilized on TALON metal affinity beads and incubated with GST-tagged Furin mutants. The upper panel shows specific staining for Mint3 from the pull-down assay, and the lower panel represents the Coomassie staining of GST-tagged Furin mutants.