Activity-Based Proteomic Profiling of Deubiquitinating Enzymes in Salmonella-Infected Macrophages Leads to Identification of Putative Function of UCH-L5 in Inflammasome Regulation
Fig 2
UCH-L5 is up-regulated while UCH-L3 is down-regulated in infected cells and both of these uncharacterized chicken DUBs are catalytically active.
(A-B). UCH-L5 and UCH-L5 DUBs are regulated in Salmonella-infected macrophages. HD11 macrophages cells were infected as mentioned above in triplicates for 0 and 18 hours with Salmonella at MOI of 50:1, followed by cell lysis. The protein samples were separated by SDS-PAGE and anti-UCH-L3 and anti-UCH-L5 western blotting was done to demonstrate changes in protein level. The anti-β actin western blot was done as a loading control (A). Alternatively, these lysates were subjected to a reaction with the Ub-VS-HA probe (B) and processed as above. (C). Overexpressed chicken UCH-L3 and UCH-L5 are active DUBs. FLAG-tagged UCH-L5 and UCH-L3 were overexpressed in HD11 macrophages and empty vector was used as a control. 24-hours past overexpression, the cells were lysed and equal amounts of proteins were incubated with Ub-VS-HA probe. The samples were analyzed by SDS-PAGE, followed by anti-FLAG western blotting to demonstrate expression and Ub-VS-HA probe binding to these DUBs. Ponceau Red staining of the membranes was used as a loading control.