FIGURE 3.

Reductions in Mon1a affect trafficking from the ER to the Golgi. A, HeLa cells stably expressing GalNAc-T2-GFP were silenced with nonspecific (NS) or human Mon1a specific oligonucleotides for 72 h. Cells were than transfected with empty vector or siRNA-resistant mouse FLAG-Mon1a and incubated for 18 h. Cells were then treated with BFA as in Fig. 1, BFA was washed away, and the movement of GalNAc-T2-GFP to the Golgi apparatus was assessed by epifluorescence microscopy. Representative images are shown from n = 5 experiments. Scale bar = 10 μm. B, NIH3T3 cells nonspecifically or Mon1a-silenced expressing ts045VSVG-GFP were grown in methionine-depleted DMEM for 2 h at 39.5 °C. Cells were then pulsed with [³⁵S]methionine for 30 min at 39.5 °C and shifted to 32 °C for the indicated times. Cells were lysed in 1% Triton X-100, 0.15 m NaCl, 10 mm Tris-HCl, pH 7.2. Lysates were immunoprecipitated for GFP, denatured, and incubated with Endo H for 2 h, and radiolabeled ts045VSVG-GFP was resolved on SDS-PAGE followed by autoradiography. A representative blot (n = 3) is presented. R, Endo H resistant; S, Endo H sensitive.