Recombinant human ephrin b2 fc chimera protein

293-GFP, 293-humanEPHB4-GFP, and 293-murineEPHB4-GFP cells were incubated with 2 μg/mL of recombinant human Ephrin B2-Fc chimera protein (R&D Systems) for 30 min on ice. These cells were washed twice using Dulbecco’s PBS (D-PBS) and further stained with APC-conjugated anti IgG-Fc antibody (BioLegend) for 30 min on ice, following which cells bound to human Ephrin B2 chimera protein were detected by flow cytometry.

Antibodies to proteins were obtained from the following sources: EphB1 (#ab129103) and phos-EphB1 (#ab61791) for Western blot were purchased from Abcam; EphB1 (#AF542) for immunohistochemistry was purchased from Novus Biologicals; Ki67, p53, CyclinA2 and E-cadherin (#3195) were from Cell Signaling Technology; β-actin (#60008-1-Ig) and GAPDH (#60004-1-Ig) were from Proteintech. Fluorescence-labeled ki67 and CD133 were purchased from Abcam, USA. Recombinant human Ephrin-B2 Fc chimera protein was purchased from R&D (7397-EB, RD Inc, MN, USA). Cisplatin (P4394) was purchased from SigmaAldrich (Sigma, MO, USA). Cells were treated with cisplatin at 10 μg/ml for A549 for 48 h.

The expression of EPHB4 was detected via phycoerythrin (PE)‐conjugated EPHB4 antibody (R&D Systems, Inc., Minneapolis, MN, USA) staining. For the detection of EPHB4‐CAR expression, transduced T cells were stained using goat anti‐Ephrin B2 antibody (R&D Systems) and then stained using PE‐conjugated anti‐goat IgG antibody (R&D Systems). Allophycocyanin (APC)‐conjugated anti‐CD3 antibody, APC‐conjugated anti‐CD8a antibody, fluorescein isothiocyanate (FITC)‐conjugated CD4 antibody, FITC‐conjugated anti‐CD45RA antibody, and APC‐conjugated anti‐CCR7 antibody (all from BioLegend) were used for the characterisation of the CAR‐T cell phenotype. To determine the binding capacity of human Ephrin B2 to cynomolgus EPHB4, 293‐human EPHB4‐GFP and 293‐cynoEPHB4‐GFP cells were incubated with the recombinant human Ephrin B2‐Fc Chimera Protein (R&D Systems) for 20 min on ice and then stained using APC‐conjugated anti‐human IgG‐Fc antibody (BioLegend). Detailed antibody information is presented in Supplementary table 2. All flow cytometry data were acquired using BD FACS Accuri C6 Plus (BD Biosciences) and analysed using the FlowJo Software (Tree Star, Inc., Ashland, OR, USA).