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. 2020 Dec 22;11:606518. doi: 10.3389/fimmu.2020.606518

Figure 4.

Figure 4

The level of αRP105-TM expression on the cell membrane is enhanced using the F2A element. (A) The middle diagram indicates the genetic construction of anti-RP105 mIgG1 bound to the transmembrane (TM). The lower diagram indicates the genetic construction of anti-RP105 mIgG1-TM bound to anti-RP105 kappa via the F2A sequence. (B) HEK293T cells were transfected with pCADEST1-HA (A/PR8) as a control of membrane protein, pCADEST1-anti-RP105 mIgG1 and pCADEST1-anti-RP105 mkappa (αRP105), or pCADEST1-anti-RP105 mIgG1-TM and pCADEST1-anti-RP105 mkappa (αRP105-TM). Two days later, the supernatants and cells were collected. Ba/F3 cells expressing RP105/MD-1 (Balk) were incubated with the supernatants, followed by incubation with APC-conjugated anti-mouse IgG (Left panel). HEK293T cells were also incubated with APC-conjugated anti-mouse IgG (Middle panel) or biotinylated anti-HA IgG1, followed by incubation with APC-conjugated streptavidin (Right panel). The expression level was analyzed using a BD FACSCanto II. (C) HEK293T cells were transfected with pCADEST1-empty (Vector), pCADEST1-HA (A/PR8), and pCADEST1-anti-RP105 mIgG1-TM and pCADEST1-anti-RP105 mkappa (αRP105-TM) or pCADEST1-anti-RP105 kappa-F2A-anti-RP105 mIgG1-TM [αRP105-TM (F2A)]. Two days later, the cells were collected and incubated with APC-conjugated anti-mouse IgG (Left panel) or biotinylated anti-HA IgG1, followed by incubation with APC-conjugated streptavidin (Right panel). The expression level was analyzed using a BD LSRFortessa. All indicated data are representative of at least two independent experiments.