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. 2020 Apr 28;9(5):1093. doi: 10.3390/cells9051093

Figure 4.

Figure 4

ONX 0914 selectively binds to i-proteasome subunits in the spleen during acute and chronic stages of viral myocarditis. Total RNA was extracted from spleen tissue of vehicle- (A) and ONX 0914- (B) treated mice sacrificed at different time points after CVB3 infection and mRNA expression levels were determined by qPCR for subunits β5, β1, and β2 (standard proteasome) as well as their i-proteasome counterparts LMP7, LMP2, and MECL-1, respectively. (C,D) Protein homogenates were obtained and subjected to Western blot analysis, showing the protein expression for the indicated catalytic subunits of the proteasome complex for three different animals per group (vehicle: C; ONX 0914: D) on days 0, 2, 8, and 28 post-infection. Analysis of β5 and MECL-1 did not yield reliable densitometric readings and their protein expression is subsequently not shown. The section of the blot corresponding to 45 kDa treated with total protein stain indicates protein loading in each lane. Protein expression (mean ± SEM) was quantified using Image Studio Lite. Relative protein expression levels normalized to baseline controls and protein loading are shown. (E) To compare efficacy of i-proteasome inhibition at the different stages of infection, protein homogenates, obtained from a representative animal of each group (vehicle- and ONX 0914 treatment, days 0, 2, 8, and 28), were loaded onto the same SDS gel and Western blot analysis was performed as indicated. Total protein stain served as the loading control. (F) Covalent binding of ONX 0914 to the catalytic subunits of the proteasome induces an upward shift of the protein band for the respective proteasome subunit, indicative of an elevation of its molecular weight by the binding of ONX 0914. Based on the shifts detected with the Western blot analysis depicted in (C) and (D), the relative inhibition achieved by ONX 0914 was calculated for each i-proteasome subunit. This was accomplished through division of the signal from the upper (ONX 0914-bound) band by the total expression signal, which was calculated by the addition of both upper and lower bands in each individual lane.