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. Author manuscript; available in PMC: 2019 Feb 1.
Published in final edited form as: J Immunol. 2017 Dec 27;200(3):928–936. doi: 10.4049/jimmunol.1700273

Figure 4. Chemical inhibition of Usp14 with 1D18 and 1B10 reduces retiree protein degradation.

Figure 4

EL4/SCARP-mCherry cells were treated with Shield-1 for 18 h and then acid-washed to remove Kb-SIINFEKL complexes and cultured without Shield-1 and in the presence of MG-132 (10 μM) or the Usp14 inhibitors IU1 (20 μM), 1D18 (5 μM), or 1B10 (5 μM) for 3 hours (A) or 2 hours (B and C). A. SCRAP-mCherry protein degradation is shown at the indicated time points and the calculated half-life for the model protein in the presence of each inhibitor is listed. B. Representative graphs demonstrating (top) the amount of precursor substrates from mCherry that contribute to (bottom) the number of Kb-SIINFEKL complexes observed in the retiree antigen presentation model. The difference in MFI of 25D-1.16 staining between cells with retired SCRAP-mCherry (labeled Shield-1) and cells treated with ethanol alone is termed the ΔKb-SIINFEKL C. Usp14 inhibitors IU1, 1D18, and 1B10 at indicated concentrations were added to cells with retired SCRAP-mCherry and the ΔKb-SIINFEKL was determined 2 h post Shield-1 removal. (*p ˂ 0.05, **p ˂ 0.01)