Fig. 3

Cloning of the 32D6 monoclonal antibody against mouse PD-1. a RT-PCR amplification of the light chain variable region (VLκ) and heavy chain variable region (VH) from the hybridoma 32D6. b Assembly of the 32D6 VH region and VLκ region through a joining fragment resulting in a 6.574-kbp product. c Schematic of the plasmid encoding the rat recombinant antibody with the FMDV 2A sequence. d Western blot analysis under reducing (left panel) or non-reducing (right panel) conditions: fresh culture medium (Mock), supernatant of cells producing the recombinant 32D6 antibody (Rec 32D6) or the 32D6 monoclonal antibody purified from the parental hybridoma (Hyb 32D6). e Immunofluorescence analysis of mPD-1 U-2 OS and U-2 OS cells with Rec 32D6 and Hyb 32D6