Figure 5.

Modulation of Trap1 expression by cytokines. (A) Trap1 (Hsp75) deficiency in DNase1^−/−/Trap1^m/m mice. Immunodetection by Western blot of mitochondrial Trap1 (Hsp75, ~74 kDa, left panel) and mitochondrial Hsp60 (~58 kDa, right panel) using splenocyte protein extracts (50 μg) of the mouse strains indicated. For loading control and for positive control, β-actin (~42 kDa) and human HeLa cell extract (50 μg) were investigated in parallel. The first lane corresponds to molecular weight markers. One representative experiment of two is shown. WT, wild type; D1^−/–/Trap1^m/m, DNase1^−/–/Trap1^m/m; D1^lacZ/lacZ, DNase1^lacZ/lacZ; kDa, kiloDalton. (B) Comparative analysis (left) of ex vivo Trap1 and β-actin expression in a series of splenocytes from the three mouse strains as in (A) with 25 μg protein extracts (top) and quantification of Trap1 expression after normalization to β-actin expression (bottom). Numbers indicate individual mice. Three mice were analyzed per genotype. As a positive control (right panel), recombinant Trap1 was loaded in parallel to protein extract from a wild type mouse and Trap1 (top) and β-actin (bottom) were immunodetected (lanes were extracted from the same Western blot but at different chemiluminescence exposure times). M, molecular weight markers. One representative experiment of two is shown. (C) Pooled data of Trap1 expression normalized to β-actin expression in splenocytes from eight independent wild type vs. six DNase1^lacZ/lacZ mice (25 μg protein extract were loaded for each mouse). n.s., not significant. Mean and SEM are shown. (D) In vitro down-regulation of Trap1 expression by cytokines. Splenocytes where cultured for 24 h in the presence/absence of IFN-γ (left) or IL-6 (right) and Trap1 expression was determined as in (C). Shown are pooled data from three independent experiments with splenocytes from eight independent mice. **p < 0.01; *p ≤ 0.05 (two-tailed Wilcoxon matched-pairs signed rank test).