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. 2020 Oct 22;29(2):718–733. doi: 10.1016/j.ymthe.2020.10.019

Figure 1.

Figure 1

Ectopic Expression of K3 and K5 in T Cells

(A) K3 and K5 are transmembrane E3 ubiquitin ligases that target MHC proteins for polyubiquitination and endocytic trafficking to the lysosome. N, N terminus; C, C terminus of K3 or K5 protein. (B) Schematic retroviral vector designs: K3.myc-Qstalk encoding K3 fused with a myc tag, and Qstalk with the transgenes separated by P2A ribosomal skipping sequences; K3.myc encoding only K3 with a myc tag. Similar vectors were prepared for K5 expression. Qstalk only was generated as the control. (C) K3 and K5 expression in transduced T cells 9 days after transduction was probed with anti-myc antibody on an immunoblot. NT, not transduced. (D) Flow cytometry analysis to determine transduction efficiency using anti-CD34 and anti-CD3 antibodies 9 days after transduction. (E) At day 10 post-transduction, K3.myc-Qstalk- and K5.myc-Qstalk-modified T cells were subjected to magnetic sorting using anti-CD34 microbeads. Flow cytometry analysis to test the frequency of the CD34+CD3+ population post-selection is displayed.