Objective      To identify Rab40b and Rab40c interacting proteins in dendritic cells with improved tandem affinity purification strategy.        Method      Vectors expressing eXact-6×His-EYFP-Rab40b or eXact-6×His-EYFP-Rab40c were constructed by molecular cloning techniques. Stable dendritic cell lines (DC2.4) expressing eXact-6×His-EYFP-Rab40b protein and eXact-6×His-EYFP-Rab40c protein were constructed by high-titer lentiviral transduction. Tandem affinity purification strategy was improved to use eXact-6×His tag to perform tandem purification and qTOF liquid mass spectrometry. Rab40b and Rab40c interacting proteins were screened by unbiased comparative metrics-based algorithm (CompPASS) and gene ontology enrichment analysis. The interacting proteins were identified by eXact pull-down and co-immunoprecipitation.       Results      Stable dendritic cell lines (DC2.4) expressing eXact-6×His-Rab40b protein and eXact-6×His-Rab40c protein were constructed respectively. Twenty-six high-confidence Rab40b interacting proteins and 16 high-confidence Rab40c interacting proteins were identified by improved tandem affinity purification. Rab40b and Rab40c interacted with ElonginB

ElonginC and Cullin5.       Conclusion      The tandem affinity purification strategy is improved successfully. ElonginB

ElonginC and Cullin5 are interacting proteins of Rab40b and Rab40c.