Figure 5. Stable, dimeric ERdj3^QEVV shows reduced binding to BiP and destabilized substrates.

1. Representative immunoblot for inputs and anti‐FLAG immunoprecipitations (IPs) of lysates prepared from HEK293T cells overexpressing ^FTBiP and the indicated ERdj3 mutant. Mock‐transfected HEK293T cells are included as a control. Cells were treated with the cell‐permeable, reversible crosslinker DSP prior to lysis to immortalize transient ^FTBiP‐ERdj3 interactions.
2. Bar graph depicting quantification of ERdj3 mutants co‐immunopurifying with ^FTBiP from immunoblots as shown in (A). Data are shown normalized to ERdj3^WT. Error bars represent standard error for n = 3. ***P < 0.005 from a paired t‐test.
3. Representative immunoblots for inputs and anti‐FLAG immunoprecipitations (IPs) of lysates prepared from HEK293T cells co‐overexpressing ^FTTTR^D18G and the indicated ERdj3 mutant. Mock‐transfected HEK293T cells are included as a control. Cells were treated with the cell‐permeable, reversible crosslinker DSP prior to lysis to immortalize transient ^FTTTR^D18G‐ERdj3 interactions.
4. Bar graph depicting quantification of ERdj3 mutants co‐immunopurifying with ^FTTTR^D18G from immunoblots as shown in (C). Data are shown normalized to ERdj3^WT. Error bars represent standard error for n = 3. *P < 0.05. **P < 0.01 from a paired t‐test.
5. Representative immunoblot of secreted ERdj3^WT or ERdj3^QEVV from conditioned media collected from transfected HEK293T cells purified using an affinity Sepharose resin conjugated to native or denatured RNase A. Conditioned media containing ERdj3^WT or ERdj3^QEVV are shown as input.
6. Bar graph depicting quantification of secreted ERdj3^WT and ERdj3^QEVV mutants associated with denatured RNase A beads as in (E). Data are normalized to ERdj3^WT. Error bars represent standard error for n = 3. *P < 0.05 from a paired t‐test.

Source data are available online for this figure.