rabbit anti l3  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: Activity-Based Proteomic Profiling of Deubiquitinating Enzymes in Salmonella -Infected Macrophages Leads to Identification of Putative Function of UCH-L5 in Inflammasome Regulation

doi: 10.1371/journal.pone.0135531

Figure Lengend Snippet: (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.

Article Snippet: The antibodies used in this study were the following: goat anti-UCH-L5 antibody (Santa Cruz Biotechnology, USA), mouse anti-β-actin antibody (Sigma-Aldrich, USA), Mouse anti-HA antibody (Sigma- Aldrich, USA), mouse anti-GAPDH antibody (Thermo Scientific, USA), mouse anti-FLAG M2 antibody (Sigma Aldrich, USA), rabbit anti-UCH-L3 antibody (Cell Signaling, USA), rabbit anti-IL-1β (Abcam, USA), rabbit anti-IL-1β (Santa Cruz Biotechnology, USA).

Techniques: Infection, Lysis, SDS Page, Western Blot, Control, Plasmid Preparation, Over Expression, Incubation, Expressing, Binding Assay, Staining