Fig. 5. Intracellular Cx43/ß-gal is in an early secretory compartment. 43ß2 cells were incubated in either the absence (A-D) or presence (E-H) of 5 µg/ml brefeldin A (BFA) for either 5 minutes (E,F) or 30 minutes (G,H) to collapse the cis and medial aspects of the Golgi apparatus into the ER. (A,B,E,F) The cells were then fixed, permeabilized and double-label immunostained using mouse anti-MG160 (A,E) and rabbit anti-ß-gal (B,F), which were detected using Texas Red-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG as secondary antibodies, respectively. Arrowheads indicate perinuclear regions where there was good colocalization between MG160 and Cx43/ß-gal (A,B). Note that both MG160 and Cx43/ß-gal were translocated to the ER by BFA treatment, as revealed by an increase in nuclear membrane (E,F, arrows) and peripheral labeling. (C,G) 43ß2 cells (C) treated to preferentially label the TGN with C₆-NBD-Cer (as described in Materials and Methods) showed perinuclear labeling, while 43ß2 cells pretreated with BFA for 30 minutes (G) showed a characteristic TGN labeling pattern (arrowheads). (D,H) In a parallel set of cells, treatment for 30 minutes with BFA (H) did not cause Cx43/ß-gal to accumulate in a condensed TGN structure. Bar, 20 µm.