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Fig. 2 Cloning of the 5F11 monoclonal antibody. (a) Reverse transcriptase-polymerase chain reaction (PCR) amplification of the variable light (VLκ) and heavy (VH) antibody chains from the 5F11 hybridoma. (b) Assembly of the 5F11 VLκ and VH regions by fusion PCR through a joining fragment resulting in a 2.4 kbp product. (c) Schematic of the plasmid encoding the recombinant antibody with the fusion PCR product cloned between NotI and AscI. (d) Western blot run under reducing (left panel) or non-reducing (right panel) conditions: tissue culture medium (Med), supernatant of cells producing the recombinant 5F11 antibody (Rec) or tissue culture medium spiked with the 5F11 monoclonal antibody purified from the parent hybridoma (Hyb). Staining of the recombinant 5F11 [Rec 5F11, (e)] is indistinguishable from that of the parent hybridoma [Hyb 5F11, (f)], red. Nuclei are counterstained in blue (DAPI). Abbreviations: cns = central nervous system; e = eye; CMV = cytomegalovirus promoter; L = leader sequence; C = constant region; V = variable region.