Fig. 1. The KDEL sequence of PDIA6 is mandatory for its cell surface association. (A) Schematic drawing of the C-terminal-tagged PDIA6. The PC-tag was inserted directly in front of the KDEL sequence. (B) Representation of the N-terminal myc-tagged PDIA6. The mature sequence of the isomerases was cloned directly behind the coding sequence of the signal peptide of the IL-6R followed by a myc-tag. (C) Western blot of cleared cell culture supernatant of HEK293T cells and the corresponding lysates, after overexpression of PDIA6 and PDIA6wo, either untagged or myc-tagged. In contrast to wild-type PDIA6, PDIA6wo is secreted into the supernatant. (D) This observation was verified by using either N-terminally myc-tagged or C-terminally PC-tagged isomerase. After overexpression of the indicated proteins, the supernatants were harvested, cleared, and divided into two halves; Protein G beads were added to both halves, but the antibody only to one half (+ab). The tagged PDIs were precipitated by using antibodies against the respective tags. In the Western blots, PDIA6 was detected by a rabbit α-PDIA6 antibody. (E) The KDEL sequence of PDIA6 is needed for its cell surface association. PDIA6 and PDIA6wo were overexpressed in HEK293T cells. After cell surface biotinylation, the biotinylated proteins were precipitated and analyzed by Western blot with antibodies against PDIA6 and b-actin. (F) Input controls of representative cell surface biotinylation experiment shown in (E). (G) Likewise, N-terminal myc-tagged and C-terminal PC-tagged PDIA6 and PDIA6wo were overexpressed in HEK293T cells. Upon biotinylation of proteins at the cell surface, biotinylated proteins were precipitated and analyzed by Western blot by using α-PDIA6 antibodies and myc-tag- and PC-tag-specific antibodies. As negative control function, β-actin was used, which was detected only in the control lysates. (H) Input controls of the lysates of cell surface biotinylation experiments.