Figure 7.

COPII recruitment is not blocked in cells lacking Yip1A. (A–F) Cells cotransfected with Sec13-Myc and either a control siRNA (A–C) or Yip1A siRNA-2 (D–F) were fixed 72 h later and doubly stained with antibodies against the Myc epitope (A and D) and calnexin (B and E). The merge is also shown (C and F). Arrows (D–F) indicate the lack of Sec13-Myc in ER whorls. Bar, 10 μm. (G–K) Cytosol dependent COPII assembly does not require Yip1A. Cells doubly transfected with a control (G and H) or Yip1A siRNA-2 (I and J) were permeabilized with 30 μg/ml digitonin 72 h after the second transfection and incubated with an ATP-regenerating system and guanosine 5′-O-(3-thio)triphosphate in the absence (G and I) or presence (H and J) of 4 mg/ml rat liver cytosol. After 20 min at 37°C, cells were fixed and stained with antibodies against Sec13. Bar, 10 μm. (K) Quantitation of the total fluorescence in Sec13-positive structures under the indicated conditions is shown, three independent experiments, ±SD.