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. 2015 Jun 23;11(9):1484–1498. doi: 10.1080/15548627.2015.1063763

Figure 1.

Figure 1

(See previous page). PARL ablation leads to mitochondrial PARK2 recruitment. (A) Endogenous PINK1–66 (white triangle) accumulated upon PARL knockdown. In order to detect robust PINK1 levels, mitochondrial membrane proteins were enriched by subcellular fractionation and sodium carbonate extraction. Mitochondrial reference proteins such as VDAC (voltage-dependent anion channel) and AIFM (apoptosis-inducing factor, mitochondrion-associated) were not affected by PARL knockdown. As control, PINK1–66 accumulation was induced by CCCP for 3 h. (B) Experimental outline to analyze the influence of PARL ablation on mitochondrial PARK2 recruitment. PARL knockdown in HEK293 T-REx-shRNA cells was induced by doxycycline as indicated. Cells were transfected with HA-PARK2 36 h before harvesting. As control, cells were treated with CCCP for 3 h. (C) PARK2 accumulated in total cell extracts and was recruited to mitochondria upon doxycycline (dox)-induced PARL knockdown (left panel). This effect was specific, since PARK2 did not accumulate in non-shRNA-expressing parental HEK293 T-REx cells treated with doxycycline for the indicated time (right panel). As control, PARK2 recruitment was induced by CCCP. IRES-GFP and the cellular markers AIFM and ACTA/actin were used as transfection and loading control, respectively. (D) Upon PARL knockdown for 14 d (+ shRNA), GFP-PARK2 colocalized with the mitochondrial marker mito-mCherry similarly, but less pronounced as observed in cells treated with CCCP for 3 h. Scale bars: 10 µm. (E) PINK1 was activated upon PARL knockdown leading to its autophosphorylation at threonine 257 (pThr257), as assessed by immunoprecipitation (IP) and western blot (WB) analysis of ectopically expressed PINK1-FLAG from the detergent solubilized mitochondrial membrane fraction. Asterisks, cross-reacting immunoglobulin heavy chain. (F) FACS-based JC-1 assay shows that doxycycline-induced PARL knockdown for 3 and 14 d (d) did not influence the mitochondrial membrane potential in HEK293 T-REx-shRNA cells. Healthy cells showed red and green stained mitochondria, whereas cells with dissipated membrane potential caused by CCCP were predominantly green. FSC, forward scatter; SSC, side scatter.