(A) Multiple sequence alignment of the predicted transmembrane domain of EMC1 from human (H. sapiens), chimpanzee (P. troglodytes), mouse (M. musculus), rat (R. norvegicus), fly (D. melanogaster), haploid yeast (C. glabrata), and baker’s yeast (S. cerevisiae). Highlighted amino acids (in green rectangles) represent highly conserved charged or polar residues. (B) CV-1 cells expressing the indicated constructs were stained with an anti-FLAG antibody. BAP31 was used as an ER marker. DAPI positions the nucleus. (C) WT EMC1*-FLAG, EMC1 (D961A)-FLAG, or EMC1 (T976A)-FLAG expressing cells were lysed and treated with endoglycosidase H followed by SDS-PAGE and immunoblotting using a FLAG antibody. (D) Cell lysates generated from HEK 293T cells expressing the indicated constructs were subjected to immunoprecipitation using anti-FLAG antibody coated beads. The bound samples were analyzed by immunoblotting using anti-FLAG and anti-S antibodies. (E) CV-1 cells were transfected with scrambled or the indicated siRNA for 24 hr prior to transfection with the indicated FLAG-tagged constructs for 24 hr. Cells were then infected with SV40 (MOI ~0.5) for 20 hr, fixed, and stained with anti-FLAG and anti-large T antigen antibodies. The percentages of T antigen positive cells were determined only in FLAG-expressing cells by using immunofluorescence microscopy. Values represent means ± SD from three independent experiments. (F) COS-7 cells were transfected with the indicated constructs for 48 hr prior to cell lysis using a buffer containing 1% DBC followed by immunoprecipitation with anti-FLAG antibody. The bound materials were analyzed by immunoblotting using the indicated antibodies. (G) As in E, except cells expressing EMC1 (1-961)-FLAG were also analyzed. (H) As in B, except EMC1 (1-961)-FLAG was used. WT EMC1*-FLAG represents siRNA-resistant EMC1, and all EMC1 mutants were generated using this construct as the template.