Figure 3. EDEM1 binding to misfolded glycoproteins does not require carbohydrates.
(A) NHK was co-expressed with blank vector (−), WT, EDEM1-FLAG single (E225Q, D370N, or E493Q) or triple (QNQ or KKK) mutants into 293T cells. Cells were radiolabeled for 15 min and chased for the indicated times. NHK and EDEM1 were isolated from the cell lysates with A1AT (α-A1AT) and FLAG (α-FLAG) antisera. Quantification of (B) % EDEM1 bound NHK and (C) % cellular NHK are displayed. Blank vector (white), WT EDEM1 (black), with the EDEM1 active site mutants as follows: E225Q (red), D370N (blue), E493Q (orange), QNQ (green) and KKK (purple) are designated in the bar graph. (D) NHKNOG was co-expressed with either blank vector (−) or WT EDEM1 in 293T cells. NHKNOG and EDEM1 were analyzed similar to A. Secreted NHKNOG was isolated from the cell culture media using A1AT antiserum. Quantification of (E) % EDEM1 bound NHKNOG and (F) % cellular NHKnog are displayed.