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. 2014 Oct 31;34(1):4–19. doi: 10.15252/embj.201488896

Figure 5. ERdj3 is co-secreted with destabilized proteins through the secretory pathway.

Figure 5

  1. A Schematic describing the co-incubation and co-expression experiments used to demonstrate ERdj3 co-secretion with destabilized TTR. For co-incubation, HEK293T cells overexpressing FLAG-tag TTR (FTTTR) variants were seeded with an equal number of cells overexpressing ERdj3WT. For co-expression experiments, HEK293T cells are transfected with both FTTTR and ERdj3WT. FTTTR was immunoisolated with M2 anti-FLAG beads from media conditioned overnight on the cells, eluted, and separated by SDS-PAGE for immunoblotting.
  2. B Immunoblot of M2 anti-FLAG immunopurifications from the conditioned media collected from HEK293T cells overexpressing FTTTRWT, FTTTRA25T, and/or ERdj3WT either as a co-incubation or co-expression experiment as shown in (A). Beads were washed in RIPA buffer prior to elution. Media inputs (1:400) are shown as a control.
  3. C Immunoblot of M2 anti-FLAG immunopurifications from media collected from HEK293T cells overexpressing ERdj3WT and FTTTR variants as indicated. Media inputs (1:400) are shown as a control.
  4. D Immunoblot of M2 anti-FLAG immunopurifications from conditioned media of HEK293T cells overexpressing FTTTRA25T and ERdj3WT in the presence of the indicated concentrations of Tafamidis, a kinetic stabilizer of TTR tetramers. Media inputs (1:400) are shown as a control.
  5. E Immunoblot of 6E10 anti-APP immunopurifications from the conditioned media of CHO cells or CHO-derived APP751-expressing (7WD10) cells overexpressing ERdj3WT as indicated. Cells were replated together as indicated 24 h after transfection at equal stoichiometry, and media conditioned for 36 h. In this case, ERdj3WT-transfected CHO cells replated with mock-transfected 7WD10 cells serves as the co-incubation condition, while mock-transfected CHO cells replated with ERdj3WT-transfected 7WD10 cells comprise the co-expression condition. Media inputs (1:150) are shown as a control.

Source data are available online for this figure.