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. 2018 Jan 9;9(10):9021–9029. doi: 10.18632/oncotarget.24078

Figure 1.

Figure 1

(A) Schematic of CAR constructs containing VEGFR2-specific Fn3-based antigen-recognition module. CARs obtained encompass leader sequences from either mIgk or Gaussia princeps luciferase (Gluc), VEGFR2-specific Fn3 sequence (VR2 FN3), myc epitope tag, hIgG1 spacer region (hinge-CH2-CH3 domains), CD28 region (transmembrane and signaling sequences), and CD3ζ region (transmembrane and/or signaling sequences). The vertical black line denotes the cell membrane. (B) FACS detection of VEGFR2 expression on the surface of HEK293T(VEGFR2+) cells stained with either recombinant FLAG-tagged Fn(VEGFR2)—VR2 FN3, FLAG-tagged Fn3 of irrelevant specificity—CEA FN3 [15], or left unstained. (C) Western blot detection of FnCAR expression in transduced Jurkat cells (anti-myc). (D) flow cytometry surface staining of kVR2-28z FnCAR-expressing Jurkat cells (becoming copGFP+ upon transduction) with anti-hinge (IgG-specific APC-labeled) conjugates. (E) Expression of the activation marker CD69 on CAR-Jurkat cells incubated with HEK293T(VEGFR2+) target cells or isogenic control cells (HEK293T) for the times indicated.