Figure 3.

Different accumulation of FP-17 and -22 at ERES after a 10°C temperature block. (a and b) CV1 cells were microinjected with the cDNA coding for EGFP-17 or -22, incubated for 75 min at 37°C, and then at 10°C for 3 h. The cells were then fixed and immunostained for Sec23 with an Alexa 568–conjugated secondary antibody. Each row shows the same field visualized for GFP (left) or Sec23 (middle), as well as the merged image with the indicated pseudocolors (right). b shows enlargements of the boxed areas in a. Arrows in b indicate Sec23-positive puncta from which FP-17 appears excluded or in which FP-22 appears concentrated. Bars: (a) 10 μm; (b) 2 μm. (c) Percentage of Sec23-positive structures enriched in FP-17, FP-22, or VSVG-GFP before and after the 10°C temperature block. The analysis was performed on cells like the ones illustrated in a and b. Averages + SD are shown (FP-17 37°C, n = 9 cells; FP-17 10°C, n = 12 cells; FP-22 37°C, n = 7 cells; FP-22 10°C, n = 12 cells; VSVG 39°C, n = 7 cells; VSVG 10°C, n = 6 cells). White and black columns refer, respectively to cells kept at 37 (FP-17 and -22) or 39°C (VSVG-GFP) for 75 min after microinjection and to cells incubated for a further 3 h at 10°C. The values were determined by manual counting (see Materials and methods). **, P < 0.0001 (determined by one way analysis of variance followed by Fischer's protected least square difference analysis). (d) Subtraction analysis of cells after the 10°C block. FI of Sec23 was determined after subtraction of the fluorescence of the indicated protein. Normalized ΔFI is the ratio of the mean intensities of the subtracted to the unsubtracted image (see Materials and methods). A lower normalized ΔFI indicates a higher degree of colocalization. Means + SD are shown (FP-17, n = 16; FP-22, n = 13; VSVG, n = 9). *, P < 0.05 (for the difference between FP-22 and FP-17 and between FP-22 and VSVG, determined by the Kruskal-Wallis nonparametric test followed by Dunn's post-analysis.