Fig. 2

Specific binding of FAP1V2 to PD-L1 and VEGFR2 led to efficient inhibition of tumor progress. A CLSM and Western blot analysis of transient expression of the intrabody genes in HeLa. B Co-immunoprecipitation assay of the dual-specificity of FAP1V2 against PD-L1 and VEGFR2. In the left panel, the schematic diagram was shown. In the right panel, PD-L1 and VEGFR2 in the input and co-immunoprecipitated components were analyzed by Western blot. The MW of PD-L1 was 36 kDa, and the MW of VEGFR2 was 152 kDa. The MW of EGFP was 29 kDa. C Schematic diagram and (D) flow cytometry assay showed that the blockage of cell surficial PD-L1 by FAP1V2 caused the reduced activity of PD-L1 to bind commercial antibodies. The cells were not treated with Triton. E Schematic diagram and (F) flow cytometry assay showed that the blockage of cell surficial VEGFR2 by FAP1V2 caused reduced activity of VEGFR2 to bind commercial antibodies. The cells were not treated with Triton. G Schematic diagram and (H) CLSM analysis showed that the blockage of intracellular PD-L1 by FAP1V2 caused reduced activity of PD-L1 to bind commercial antibodies. The cells were treated with 0.1% Triton. I Schematic diagram and (J) CLSM analysis showed that the blockage of intracellular VEGFR2 by FAP1V2 caused reduced activity of VEGFR2 to bind commercial antibodies. The cells were treated with 0.1% Triton. For all the above experiment, the red fluorescence showed RBITC-labeled commercial antibody specific for PD-L1 or VEGFR2, and the blue fluorescence showed cell nuclei stained by DAPI. The ability of PD-L1 and VEGFR2 to bind the commercial antibodies was determined after transient transfection of HeLa cells with plasmids pEGFP-C1, pEGFP-C1-AP1, pEGFP-C1-AV2 or pEGFP-C1-AP1V2, respectively