Fig. 5. Identification and verification of H4S1p interacting proteins.

(a) Identification of H4S1ph interacting protein by LC-MS/MS. Pull-down product were separated by gel electrophoresis, SYPRO Ruby-stained, and excised as shown in (b) for in-gel tryptic digestion. The bands of interest were not recognizable on the SYPRO^® Ruby-stained gel, so excision of gels were performed by estimating the position from molecular weight markers. Identified proteins are shown in the table. (c) Immunodetection of crosslinked product using pan-14-3-3 antibody. Asterisks indicates crosslink-product of 14-3-3 proteins and Dz-H4S1ph-biotin. (d) Specific inhibition of the interaction between H4S1ph and 14-3-3 by R18 peptide (10 μM).