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. 2014 Dec 1;33(6):401–408. doi: 10.1089/mab.2014.0043

FIG. 2.

FIG. 2.

Epitope mapping of CREPT MAb 3E10. (A) HEK293T cells were transfected with Flag-tagged CREPT or control vector. Cell lysates were blotted with MAb 3E10 and then, after stripping the membrane, with an anti-Flag antibody. (B) HEK293T cells were transfected with Myc-tagged CREPT and Myc-tagged p15RS. Western blot was performed using the indicated antibody. No cross-reaction was observed. (C) Random clones from the whole library were aligned to full-length CREPT sequence. (D) Positive clones binding 3E10 were enriched by a sorting process. (E) Alignment of positive clones. Green lines represent the forward sequences and red lines represent reverse ones. The overlapping nucleotide sequences of positive yeast clones were selected and aligned to full-length CREPT sequence (marked in red box). The below sequence of 160–168 amino acids, corresponding to the red box, was shown for the comparison with the sequence of p15RS. The sequences of the epitope in different animals are shown. (F) HEK293T cells were transfected with Flag-tagged full-length RPR domain and CCT domain of CREPT. Cell lysates were detected with either an anti-Flag antibody or monoclonal antibody 3E10.