Fig 1: Peptide aptamer candidates targeting the interaction interface on the N-terminus of CDP protein are screened from a peptide aptamer library.A Four candidate peptide aptamers targeting the interaction interface on CDP protein were screened using BiFc after cotransfection of pBiFc-VN173-CDPΔC (101-1516) with plasmids from the pBiFc-VC155-TrxA-peptide-TrxA library. Scale bar: 50 μm. B Interactions between Flag-fused four candidate peptide aptamers and the 100-residue N-terminal of CDP protein was shown by immunoprecipitation after cotransfection of pCMV-Tag2B-peptide and pBiFcVC155-CDPΔC (101-1516), respectively. C Nucleotide sequences coding four candidate peptide aptamers and amino acid sequences.
Fig 2: CDP protein levels are markedly increased in ESCC clinical samples and cell lines.A High levels of CDP protein in ESCC clinical samples, shown by immunostaining on a tissue chip. C: Cancer; CA: Cancer adjacent normal tissues. B Representative figure showing CDP protein expression in an ESCC sample and its matched normal tissue. C Significantly higher levels of CDP protein in ESCC tumor tissues (n = 75, p < 0.001). D Significantly higher levels of CDP protein in the cytoplasm of ESCC tumor tissues (n = 75, p < 0.001). E Significantly higher levels of CDP protein in the nuclei of ESCC tumor tissues (n = 75, p < 0.001). F Percent of pathological stage in ESCC clinical samples when the correlation index of CDP and SOX2 proteins reached 1. G CUX1 mRNA levels in ESCA tumor samples, determined in 182 ESCA tissues and 286 normal tissue samples using GEPIA (p < 0.05). H Abundant expression of both CDP and SOX2 proteins in ESCC cells. I Colocalization of SOX2 and CDP proteins in multiple esophageal cell lines. Scale bar: 20 μm. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. The data represent the means ± SDs.
Fig 3: The interaction interface between SOX2 and CDP protein is determined by Duolink PLA, BiFc and immunoprecipitation assays.A Interaction between CDP and SOX2 protein was analyzed by Duolink PLA in KYSE450 cells and KYSE30 cells. PLA signals were detected by fluorescence microscopy and determined by discrete red spots. A single SOX2 antibody alone or a single CDP antibody alone in KYSE450 cells was used as a negative control. B Multiple constructs expressing full-length CDP protein and SOX2 protein or their respective truncated mutants were used for the BiFc assay. C Interactions between CDP protein, SOX2 protein, and their respective mutants were verified by BiFc. Scale bar: 50 μm. D The interacting interface on the CDP protein and SOX2 protein were explored with a cotransfection BiFc assay. Note that the interaction between the 1-100 N-terminal CDP sequence and the 278-317 sequence of SOX2 was verified by BiFc assay. E Confirmation of the interaction between CDP protein mutants and SOX2 protein by immunoprecipitation.
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