Objective To screen the molecules interacting with deubiquitinase cylindromatosis(CYLD)in inflammatory response,and to explore and verify the intracellular interaction between CYLD and bridge protein 14-3-3 family molecules involved in the regulation of NF-κB signaling pathway.Methods Molecules that might interact with CYLD were screened by co-immunoprecipitation(Co-IP)and flight mass spectrometry.Under different stimulation conditions,bone marrow primary macrophages were induced to polarize towards M1 and M2.qRT-PCR and Western blotting were used to detect the expression levels of CYLD and candidate molecule 14-3-3 that might interact with CYLD in macrophages.Results CYLD antibody could capture all seven subtypes of 14-3-3 protein(β,γ,ε,η,ζ,δ,and θ),suggesting that CYLD may interact with 14-3-3 protein.The three subtypes of 14-3-3(14-3-3γ/ε/η)with the most specific peptide segments were selected from the mass spectrometry.The results showed that CYLD and 14-3-3γ/ε/η were expressed in M0,M1 and M2 macrophages.Furthermore,Myc-CYLD and Flag-14-3-3γ/ε/η eukaryotic overexpression vectors were constructed,and CYLD protein was precipitated by Flag antibody.Co-IP results indicated that CYLD could interact with 14-3-3γ/ε/η.Immunofluorescence assay showed that CYLD and 14-3-3 were co-localized in macrophages.Conclusion There may be physical interaction between CYLD and 14-3-3-γ/ε/η in inflammatory response of macrophage.
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