Fig. 6. Effect of Rab9S21N and thapsigargin (Thaps) on the sulfation of M6PR46-HMY and ricin sulf-2. (A,B) MDCK II cells transfected with M6PR46-HMY or contransfected with M6PR46-HMY and Rab9S21N were preincubated for 30 minutes at 37°C with or without 0.1 µg/ml thapsigargin. Then radioactive sulfate was added, and the incubation was continued for 3 hours. The cells were subsequently washed, lysed and immunoprecipitated using Ni-agarose beads. The adsorbed material was eluted with 25 mM EDTA and analyzed by SDS-PAGE (12%) before autoradiography. (C) Graphic illustration of the signal intensities of the bands in B representing sulfated M6PR46-HMY. The band intensities were determined by densitometric quantification using Image-Quant 5.0. (D) MDCK II cells untransfected or transfected with Rab9S21N were incubated with radioactive sulfate for 3 hours at 37°C, and thapsigargin (0.1 µg/ml) was added for the last 30 minutes. Then ricin sulf-2 (200 ng/ml) was added to the medium, and the cells were further treated as described in the legend to Fig. 3.