Fig. 4

CGN c.3560C > T leads to overexpressed IQGAP1 identified by proteomic analysis. A Proteomic analysis using two-dimensional (2D) gel electrophoresis was conducted on lysates from HT-29 cells with either CGN c.3560 C > C or c.3560 C > T, both with and without EGF treatment over a 48-hour period. Enlarged images of specific 2D gel spots, denoted by arrows, were employed to underscore the observed variations in expression levels. A gel spot (solid circle) from CGN c.3560 C > T treat with EGF group was selected and followed by LC-MS/ MS analysis. B (Upper) Assignments to a protein in the searched database are established by correlating mass-to-charge ratio (m/z) values derived from both the Peptide Mass Fingerprinting (PMF) and the tandem mass spectrometry (MS/MS) data of the peptide ILAIGLINEALDEGDAQK. (Bottom) The peptide mapping figure and the identified sequence correspond to the protein IQGAP1. C The specific interaction of GFP-CGN WT, GFP-CGN c.3560 C > T, and IQGAP1 in HT-29 cells was investigated through immunoprecipitation (IP) using a GFP antibody. IP lane represents immunoprecipitation of bound eluted proteins, and input lane represents whole cell lysate. IP were analyzed by Western blotting using anti-IQGAP1 and anti-GFP antibody, and CGN expression in input was using anti-CGN antibody