Table 1.

Quantitation of Antibody Colocalization in Confocal Immunofluorescence Images

Antibodies Compared	Percentage of Colocalization (Mean ± sd)	n
VSR antibodies versus AtSYP21 versus 14G7 antibodies (Figure 2)
VSR:CT and VSR:14G7	94 ± 6	12
VSR:AtSYP21 and CT:AtSYP21	91 ± 6	15
YFP reporters versus anti-GFP in transgenic BY-2 cells (Figure 3)
GONST1-YFP:anti-GFP	93 ± 6	23
YFP-BP-80:anti-GFP	96 ± 3	18
YFP reporters versus anti-VSR in transgenic BY-2 cells (Figure 5, panels 1 and 2)
YFP-BP-80:anti-VSR	97 ± 3	9
GONST1-YFP:anti-VSR	8 ± 2	15
YFP reporters versus AtSYP21 in transgenic BY-2 cells (Figure 5, panels 3 and 4)
YFP-BP-80:anti-AtSYP21	83 ± 2	6
GONST1-YFP:anti-AtSYP21	2 ± 3	11
YFP reporters versus anti-ManI in transgenic BY-2 cells (Figure 5, panels 5 and 6)
YFP-BP-80:anti-ManI	4 ± 3	10
GONST1-YFP:anti-ManI	91 ± 4	14
YFP reporters versus anti-VSR in transgenic BY-2 cells treated with wortmannin (33 μM) for 3 h (Figure 8C)
YFP-BP-80:anti-VSR	85 ± 5	10
YFP reporters versus FM4-64–labeled PVCs (Figure 14)
YFP-BP-80:FM4-64	85 ± 4	19
GONST1-YFP:FM4-64	11 ± 4	25

YFP reporters in transgenic BY-2 cells were studied by confocal immunofluorescence localization. Antibody marker for YFP was anti-GFP; antibody marker for Golgi was anti-ManI; and antibody marker for VSR protein and PVC was anti-VSR. FM4-64 is a fluorescent dye that labels prevacuolarment after endocytosis. Quantitation of the extent of colocalization for the two antibodies (or proteins) was performed from one direction only (i.e., to determine how much of the YFP-BP-80 reporter proteins colocalized with anti-VSR and not the other way around) as described previously (Jiang and Rogers, 1998). Percentage of colocalization is expressed as the mean ± sd for the number of cells analyzed (n). Also, a t test was performed to compare results from the individual paired assays of the two reporters expressing in BY-2 cells. The resulting P values for the comparison were statistically significant (P < 0.01).