FIGURE 1. Isolation and preparation of proteins for mass spectrometric analysis.

(A) Validation of nuclear extraction from TSU-Pr1 cells stably expressing KLF5 (K12 clone). The lamin A/C nuclear marker and β-tubulin cytoplasmic marker were detected by western blotting. (B) Silver staining gel analysis of KLF5 interacting proteins pulled down using KLF5 antibody by co-IP (about 1/10 of the elution from co-IP) in K12 cells. Proteins pulled down using purified IgG served as a negative control. Bands for the light and heavy chains of IgG are indicated by asterisks. For mass spectrometry, four fifths of the co-IP elution for each KLF5 or the IgG negative control were concentrated, separated in another gel and stained with Coomassie blue, and both the light and heavy chain bands of IgG were excised and discarded to reduce nonspecific readings. The remaining gel of the entire lane, as indicated dotted red lines, was collected and subjected to mass spectrometry. (C) Confirmation of the presence of KLF5, c-Jun, and CINP in the co-IP products by western blotting. c-Jun, a known KLF5 interacting protein, was used as a positive control. β-actin was used as a negative control. (D) Top candidates of KLF5 interacting proteins identified by co-IP and mass spectrometry analysis. ¹ Score: Protein scores, calculated by Proteome Discoverer application from a list of peptides identified for a particular protein, indicate the relevance of a protein. ²Coverage: Coverage of identified high-confidence peptides match the protein. ³Peptides: Number of high-confidence peptides which match the protein.