Immunofluorescence using anti-58K protein antibody. (a1�Ca4) Focal series (2�C8 ��m from the base of the cells) showing the distribution chemical information of Golgi elements … Our investigation of the Golgi complex was complemented by determining the distribution of the ERGIC and TGN. In polarized CFPAC-1 cells, ERGIC-53 and ��-adaptin immunoreactivity had the same distribution as that for 58K protein. Figures 4a and and4b4b show the dispersal in the perinuclear cytoplasm of the ERGIC and TGN, respectively. In polarized CFPAC-PLJ-CFTR6 cells, these compartments were clustered in a perinuclear or supranuclear cytoplasmic region (Figures 4c and and4d,4d, arrows). Figure 4. Distribution of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) (a,c) and trans-Golgi network (TGN) (b,d) in CFPAC-1 (a,b) and CFPAC-PLJ-CFTR6 (c,d) cells.

Immunofluorescence using anti-ERGIC-53 (a,c) and anti-��-adaptin (b,d) … We used double labeling to localize CA IV in the different Golgi compartments. A dispersal of CA IV immunoreactivity (Figure 5a) similar to that for 58K protein (Figure 5b) was observed in polarized CFPAC-1 cells. Superimposing these two images showed that CA IV was clearly localized at the Golgi complex (Figure 5c). In polarized CFPAC-PLJ-CFTR6 cells, double labeling for CA IV (Figure 5d) and 58K protein (Figure 5e) indicated their colocalization in the supranuclear cytoplasm (Figure 5f). We also observed the colocalization of CA IV with ERGIC-53 and ��-adaptin in the two cell lines. Figure 5. Demonstration of CA IV in the Golgi complex of CFPAC-1 (a�Cc) and CFPAC-PLJ-CFTR6 (d�Cf) cells.

CA IV/58K protein double labeling. (a,d) CA IV immunoreactivity. (b,e) 58K protein immunoreactivity. (c,f) Superimposition of 58K protein and … In CFPAC-PLJ6 cells, we obtained results identical to those for CFPAC-1 cells. Microtubules and the Golgi Complex Confocal microscopic examination of ��-tubulin immunoreactivity showed differences in the distribution of microtubules between CFPAC-1 and CFPAC-PLJ-CFTR6 cells. In polarized CFPAC-1 cells, focal planes passing through the basal and medial cytoplasms displayed a network of dispersed microtubules that extended from the perinuclear regions to the periphery of cells (Figure 6a 1). In polarized CFPAC-PLJ-CFTR6 cells, focal planes passing through the medial cytoplasms showed ��-tubulin immunoreactivity located along lateral plasma membranes (Figure 6b 1).

In supranuclear cytoplasms of CFPAC-1 cells, ��-tubulin immunoreactivity appeared as a network of dispersed Dacomitinib filaments radiating from different focal points (Figure 6a 2), whereas in CFPAC-PLJ-CFTR6 cells it appeared in the form of a dense, tangled, filamentous network (Figure 6b 2). The addition of nocodazole to CFPAC-1 and CFPAC-PLJ-CFTR6 cell cultures resulted in complete microtubule depolymerization.