Skip to main content
. 2020 Feb 17;9:e53455. doi: 10.7554/eLife.53455

Figure 6. Effect of mutation of various cysteine residues of TXNDC11 on its disulfide-bonding to EDEM2 and on gpERAD.

Figure 6.

(A) Structure of human TXNDC11 is schematically shown with cysteine residues (C) highlighted together with their positions. (B) Cell lysates were prepared from WT, EDEM2-KO, and TXNDC11-KO cells expressing WT or one of various cysteine mutants of 3x Flag-tagged TXNDC11 together with Myc-tagged EDEM2 by transfection, subjected to SDS-PAGE under non-reducing conditions, and analyzed by immunoblotting using anti-TXNDC11 and anti-EDEM2 antibodies. (C) Cell lysates were prepared from TXNDC11-KO cells expressing WT or one of the three cysteine mutants of 3x Flag-tagged TXNDC11, and subjected to immunoprecipitation using anti-Flag antibody. An aliquot of cell lysates (Input) and the immunoprecipitates {IP(Flag)} were subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-EDEM2 and anti-TXNDC11 antibodies. (D) Cell lysates were prepared from WT, EDEM2-KO, and TXNDC11-KO cells expressing WT or one of the three cysteine mutants of 3x Flag-tagged TXNDC11 together with mCD3-δ-ΔTM-HA by transfection, and analyzed by immunoblotting using anti-HA and anti-TXNDC11 antibodies. (E) Cycloheximide chase was conducted to determine the degradation rate of mCD3-δ-ΔTM-HA in WT and TXNDC11-KO cells expressing WT or one of the three cysteine mutants of 3x Flag-tagged TXNDC11 by transfection, and cell lysates were analyzed by immunoblotting using anti-HA antibody (n = 3). Quantified data are shown on the right.