Fig. 1. Specific inhibition of retrograde transport of STxB in Syn16 RNAi conditions. (A) Metabolic sulfation of internalized STxB-Sulf2 on HeLa cells over a 20-minute period was visualized by immunoprecipitation from cells that had been transfected for 72 hours with three different siRNAs against Syn16 or with scrambled (Sc) siRNA (200 nM each). Data are expressed as percentages of STxB-Sulf2 sulfation for each experimental condition, using sulfation of STxB-Sulf2 on mock-transfected cells as the reference (set to 100%), and normalized for total sulfation counts (see Materials and Methods and Fig. 2C). Inset: Western blotting confirmed the down-modulation of Syn16 under these conditions. Tubulin was used as a loading control. (B) Sulfation analysis (as in A) of STxB-Sulf2 applied to HeLa cells that had been transfected for 72 hours with increasing doses of anti-Syn16 siRNA number 3. Inset: Western blot of Syn16 under the indicated conditions. (C-E) Immunofluorescence analysis of retrograde STxB transport in (C) mock-transfected control (CTL) and (D-E) Syn16 RNAi cells (siRNA sequence number 3 at 200 nM). In all conditions fluorophore-labeled STxB (green) was internalized for 45 minutes at 37°C. Cells were fixed and labeled for Syn16 (blue), the Golgi marker CTR433 (red, D), or TfR (red, E). Under Syn16 RNAi conditions, STxB accumulation in the Golgi was reduced, and the protein appeared in peripheral structures where it co-distributed with the TfR. Bar, 10 µm. (F-H) Specificity controls for the Syn16 RNAi. (F) EGF degradation. 2.5 ng/ml of iodine-labeled EGF was incubated for the indicated times with HeLa cells in the indicated conditions. Lysosomal degradation was measured by appearance of TCA-soluble counts in the culture medium (G) Tf recycling. Biotin-tagged Tf was internalized at 37°C for 40 minutes into HeLa cells in the indicated conditions. Cells were washed and further incubated as shown at 37°C. Remaining cell-associated Tf was determined by ELISA. In F-G, the means of three to four independent experiments are shown. (H) VSVG transport in the biosynthetics/secretory pathway. The trafficking of newly synthesized VSVG-ts045 to the plasma membrane was analyzed by cell-surface biotinylation after a shift in temperature from 40°C to 32°C. BFA was used as a specificity control.