Figure 3. Effect of TXNDC11 knockout on EDEM2.

(A) Quantitative RT-PCR was conducted to determine the levels of endogenous EDEM2 mRNA (a) using the two primer sets indicated as well as spliced XBP1 mRNA (b) relative to the level of GAPDH mRNA in WT and two TXNDC11-KO cells (n = 3). (B) Cell lysates were prepared from WT, EDEM2-KO, and two TXNDC11-KO cells, subjected to SDS-PAGE under reducing and non-reducing conditions, and analyzed by immunoblotting using anti-EDEM2 and anti-TXNDC11 antibodies. (C) Cell lysates were prepared from WT, EDEM2-KO, and TXNDC11-KO#1 cells expressing 3x Flag-tagged TXNDC11 or both 3x Flag-tagged TXNDC11 and Myc-tagged EDEM2 by transfection, subjected to SDS-PAGE under non-reducing conditions, and analyzed by immunoblotting using anti-EDEM2 and anti-TXNDC11 antibodies. (D) Cell lysates were prepared from WT and TXNDC11-KO#1 cells, treated with the indicated amount of trypsin at 4°C for 15 min, subjected to SDS-PAGE under reducing and non-reducing conditions, and analyzed by immunoblotting using anti-EDEM2 antibody. The band with open triangle denotes a non-specific protein which serves as a control for trypsin digestion. Quantified data are shown at the bottom.

Figure 3—figure supplement 1. Construction of TXNDC11-KO cells.

(A) Strategy of the CRISPR/Cas9-based PITCh method to target exon 1 of the TXNDC11 gene is shown. (B) Structures of the TXNDC11 locus in TXNDC11-KO#1 cells are schematically shown. (C) Structures of the TXNDC11 locus in TXNDC11-KO#2 cells are schematically shown. (D) Genomic PCR was carried out to confirm recombination. Primers for lanes 1, 2 and 3 are 753Fw and 1708Rv, TgFw and 1831Rv, and 730Fw and TgRv, respectively. (E) Total RNA was prepared from WT and two TXNDC11-KO cells, and subjected to RT-PCR to amplify cDNA corresponding to full length TXNDC11 and GM130. (F) Cell lysates were prepared from WT and two TXNDC11-KO cells, and analyzed by immunoblotting using anti-TXNDC11 and β-actin antibodies.