Fig. 4. Retrograde transport of different cargo proteins in Syn5 RNAi and Syn16 RNAi cells. (A) Constructs used for sulfation analysis. A genetic fusion of STxB with tandem sulfation sites was made as described (Mallard et al., 1998). For CTxB, a tandem-sulfation-site peptide was chemically coupled to primary amines. In the case of MPR, the tandem-sulfation-site peptide was chemically coupled to an anti-GFP antibody recognizing MPR-GFP protein in stably transfected HeLa cells (Waguri et al., 2003). (B) Sulfation analysis of sulfation-site -peptide-coupled CTxB and MPR. HeLa cells were incubated for 40 minutes with the indicated proteins in the presence of radioactive sulfate with or without BFA, followed by immunoprecipitation and autoradiography. For CTxB, the arrow indicates sulfated toxin-B subunit, whereas the upper bands, visible under conditions with or without BFA, originate from contaminating endogenous sulfoproteins at the level of the load. (C,D) STxB-Sulf2, CTxB-Sulf2 and sulfation-site-coupled anti-GFP-MPR antibody were incubated for 40 minutes in the presence of radioactive sulfate with HeLa cells that had been transfected with (C) Syn16 siRNA number 3 or (D) Syn5 siRNA number 1. Sulfated proteins were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Sulfation signals are expressed as normalized percentages of sulfation observed in mock-transfected control cells.