TABLE 1.

Quantitative analysis of the colocalization between SgII domain chimeric proteins and markers of the secretory pathway in PC12 cells

Colocalization between endogenous DCG markers SgII and CgB are shown (A). Colocalization between SgII domain/GFP (B) or SgII domain/EAP (C) fusion proteins and endogenous DCG marker CgB is shown. Colocalization between SgII domain-GFP chimeras and trans-Golgi/TGN-retained CFP chimeras GFP is shown (D). xy optical sections were acquired with increments of 0.2 μm along the z axis for the indicated number of cells. The degree of colocalization between fluorescent molecules was measured on a pixel-per-pixel basis for the two wavelength sources by comparing the position of each pixel in each of the 0.2-μm xy image of a given z stack. R_p and R_o were determined using the colocalization algorithm of ImageMaster image processing software, as described under “Experimental Procedures.” Statistical significance of correlation within B, C, and D data sets was also tested by ϰ² for a 2 × 4 contingency table, using pixel counts from a representative xy optical section taken in the mid-cell region. Group B, c² = 5925, p < 0.001; group C, ϰ² = 6548, p < 0.001; group D, c² = 1642, p < 0.001.

	Pearson's coefficient (Rp)	Overlap coefficient (Ro)	xy optical sections (n)	Cells (n)
A. Endogenous SgII vs. CgB	0.76 ± 0.02	0.75 ± 0.02	254	17
B. GFP chimera vs. endogenous CgBa
SIG-GFP	0.13 ± 0.04	0.17 ± 0.04	72	4
SIG-SgII-(25-41)-GFP	0.10 ± 0.05b	0.17 ± 0.04b	71	5
SIG-SgII-(334-348)-GFP	0.39 ± 0.04c	0.44 ± 0.01c	73	4
SIG-SgII-GFP	0.39 ± 0.05c	0.41 ± 0.05c	121	6
C. EAP chimera vs. CgBd
SIG-EAP	0.19 ± 0.02	0.25 ± 0.02	85	4
SIG-SgII-(25-41)-EAP	0.52 ± 0.07c	0.60 ± 0.06e	129	6
SIG-SgII-(334-348)-EAP	0.67 ± 0.08e	0.72 ± 0.06e	97	4
SIG-SgII-EAP	0.49 ± 0.05c	0.52 ± 0.05c	95	4
D. GFP chimera vs. CFP chimeraf
SIG-SgII-GFP vs.
GalT-CFP	0.15 ± 0.06	0.17 ± 0.06	43	4
GalT-CFP-SgIIa	0.77 ± 0.03e	0.77 ± 0.03e	43	4
SIG-GFP vs.
GalT-CFP	0.73 ± 0.07e	0.74 ± 0.07e	27	3
GalT-CFP-SgII	0.64 ± 0.03e	0.65 ± 0.03e	35	3

^aValues are given as the means ± S.E. p values for difference within groups were determined as compared with the lowest value of colocalization coefficient obtained for SIG-GFP versus CgB.

^bp > 0.05; ANOVA with Dunnett's post test.

^cp < 0.01; ANOVA with Dunnett's post test.

^dp values for difference within groups were determined as compared with the lowest value of colocalization coefficient obtained for SIG-EAP versus CgB.

^ep < 0.001; ANOVA with Dunnett's post test.

^fp values for difference within groups were determined as compared with the lowest value of colocalization coefficient obtained for SIG-SgII-GFP versus GalT-CFP.