Figure 8.

Expression and localization of various mutant forms of ATF6 in transfected HeLa cells. (A) Schematic structures of ATF6 derivatives analyzed. Full-length ATF6 cDNA, ATF6 (670), and three C-terminal deletion mutants were inserted into the mammalian expression vector pCGN. The positions of the HA epitope, basic region, leucine zipper, and transmembrane domain are indicated. (B) Immunoblotting analysis of transfected cells. HeLa cells in 60-mm dishes were transiently transfected with 1 μg of pCGN vector alone (Vec) or each of the ATF6 expression plasmids as indicated. Total proteins were extracted from transfected cells directly with 1× Laemmli’s SDS sample buffer followed by boiling for 5 min. Samples were subjected to SDS-PAGE (10% gel) and analyzed by immunoblotting with anti-ATF6 antibody. (C) Indirect immunofluorescence analysis of transfected cells. HeLa cells on slide glasses transiently transfected with pCGN-ATF6 (402) (a–c), pCGN-ATF6 (373) (d–f), and pCGN-ATF6 (366) (g–i) were fixed and stained with anti-HA epitope antibody (a–g), anti-KDEL antibody (b–h), or DAPI (c–i). Bar, 10 μm.