Figure 4.

p125A colocalizes extensively with Sec31A. (A) HeLa cells were fixed and labeled with anti-p125A antibodies (a, d, and g) and DAPI (b, e, and h). For the second (d–f) and third (g–i) panels, the antibodies were preincubated with 500 µg of recombinant GST-Bet3 and GST-p125A, respectively. The merged images are shown in c, f, and i. Bar, 10 µm. (B) HeLa cells were fixed and double-labeled with rabbit anti-p125A and mouse anti-Sec31A antibodies. Enlarged images of the boxed area are shown in the bottom panels. Bar, 10 µm. (C) HeLa cells were transfected to coexpress p125A-Cherry (a) and Sec31A-GFP (b). The live cells were imaged with a confocal microscope (Fluoview 1000; Olympus). Merged picture and DIC image are shown in c and d, respectively. Bar, 10 µm. (D) p125A- and Sec31A-positive structures are colocalized stably with each other over time, and undergo homotypic fusion. HeLa cells were transfected to coexpress p125A-Cherry and Sec31A-GFP. The cells were subjected to live-cell imaging. The vesicular structures of p125A-Cherry colocalize extensively with those of Sec31A-GFP over a period of at least 30 min (Video 1). Inset is enlarged in panels shown in E. Bar, 10 µm. (E) p125A-Cherry and Sec31A-GFP containing vesicular structures undergo homotypic fusion as indicated by the boxed areas (Video 2). Bar, 2 µm.