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. 2021 Apr 1;11:7320. doi: 10.1038/s41598-021-86847-2

Figure 6.

Figure 6

Rescue of the degradation of DJ-1 unstable mutants transfected in LonP1-silenced DJ-1-null MEFs by transfection of human LonP1. LonP1 shRNA-transduced DJ-1-null MEFs (sh mLonP1) were complemented with ectopically expressed human LonP1 (hLonP1). (A) Panel shows a representative immunoblot of LonP1 shRNA-mediated knockdown in DJ-1-null MEFs and the recovery of LonP1 protein levels after transfection with hLonP1. Immunoblot was developed with specific antibodies anti-LonP1. Quantification is shown in the right graph. LonP1-silenced DJ-1-null MEFs (sh mLonP1), panel (B) and rescued LonP1-silenced DJ-1-null MEFs (sh + hLonP1), panel (C), were transiently transfected with hDJ-1 P158Δ or L166P and 48 h after transfection were treated with 25 µg/mL cycloheximide (CHX) for the times indicated. Panels (B) and (C) show representative immunoblots developed with anti DJ-1 polyclonal antibody of the indicated constructs. Anti-tubulin antibodies were used as total protein loading control. Quantifications are shown in the graphs below as mean ± s.e.m from three different experiments. Significant differences were found between sh mLonP1 and sh + hLonP1 DJ-1-null MEFs transfected with P158Δ at the time points 2 h (*p = 0.01), 4 h (*p = 0.03), 8 h (*p = 0.03) and 12 h (*p = 0.04) and between sh mLonP1 and sh + hLonP1 DJ-1-null MEFs transfected with L166P at the time points 4 h (*p = 0.01), 8 h (**p = 0.001) and 12 h (**p = 0.001).