Supporting data for "Chemical Approaches to Investigate Histone H3.3-specific Binding Proteins and Microtubule Inner Proteins"

In my first project, a potential specific binder of histone named POI was found through in vivo crosslink-assisted and SILAC-based protein identification. After the development of biotinylated peptide or nucleosome probe for pull-down experiment, POI was validated to specifically recognize hsitone in cell lysate. The SDS-PAGE data (<b>file Gel(1)-(30)</b>) was used to validate the identity and purity of the obtained POI protein, and was further applied in the study of protein-protein interactions between POI and nucleosome probe. Another important part is the validation of the interaction in the nuclear extract, the enriched proteins can be resolved on the gel, tranferred to the PVDF membrane. After the incubation with with the primary antibody followed by secondary antibody, the signal of POI can be visualized after treated with the peroxide-luminol mixture, and the data was shown in the <b>Western blot (1) to (38)</b>.In the second project, an investigation of microtubule inner proteins (MIPs) was attempted utilizing two different in vivo crosslink-assisted and SILAC-based protein identification methods based on the incorporation of the photo-affinity amino acid or on the chemical probes derived from microtubule stabilizing agents (MSA). For the first method, photo-affinity amino acid was incorporated into α or β-tubulin by Amber suppression through transient transfection. For the second method, I synthesized several diazirine-containing photo-affinity probes modified from Paclitaxel, which is a well-known drug that targets microtubule structure. Then the development and optimization of the photo-catalytic crosslinking method specially designed for the microtubule system gradually solves the problem that the UV irradiation and photo-induced cytotoxicity of the iridium catalyst easily trigger the microtubule disassembly. Taking advantage of the chemical probes located in the lumen, the proteins can be captured after photo-catalytic crosslinking and further identified based on proteomics techniques. The data (<b>NMR (1)-(57)</b>) show the identification result of the synthesized compound in the development of chemical probes.