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. 2013 Jun 27;50(6):793–804. doi: 10.1016/j.molcel.2013.05.014

Figure 6.

Figure 6

shRNA Knockdown of Endogenous ERdj5 Compromises Native Disulfide Bond Formation and Secretion of LDLR

(A) HT1080 cells were treated with either ERdj5-specific or scrambled shRNA as indicated, and the level of ERdj5 remaining after 5 days was quantified using actin as a loading control.

(B) Comparison of the level of ERdj5 in ERdj5 knockdown cells transfected with various ERdj5 constructs as indicated. Level of expression compared to nondepleted cells is as indicated.

(C) HT1080 cells were treated with scrambled shRNA for 5 days and then cotransfected with HA-LDLR and empty vector. After a further 24 hr, cells were pulse labeled for 30 min and chased for the indicated times. Radiolabelled LDLR was immunoisolated from the cell lysate with the HA antibody and analyzed under nonreducing conditions.

(D–G) HT1080 cells were treated with shRNA directed against ERdj5 then cotransfected with HA-LDLR and either empty vector (D), ERdj5 (E), ERdj5 H63Q (F), or ERdj5 AXXA mutant (G). Cells were pulse labeled for 30 min, and radiolabelled LDLR was isolated from the cell lysate and analyzed as in (C). The ER and Golgi forms are indicated as well as disulfide-bonded intermediates.