Fig 2: PINK1 promotes NF-?B activation and modulates NEMO ubiquitination Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression. SH-SY5Y cells were transfected with control or PINK1-specific siRNAs. For rescue experiments, cells were co-transfected with wildtype PINK1, PINK1?77, or kinase-dead PINK1-K/D. Two days after transfection the cells were treated with TNF (25 ng/ml, 25 min), fixed, and stained with p65 antibodies. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of three independent experiments. The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition. ***P < 0.001, Student's t-test, two-tailed, n = 3.PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co-transfected with V5-tagged wildtype PINK1 or kinase-dead PINK1-K/D and HA-tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p-S65-ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p-S65-ubiquitin, and GAPDH.Linear ubiquitination of NEMO is reduced in PINK1-deficient cells. HEK293T cells were co-transfected with HA-NEMO and control or PINK1-specific siRNAs. Forty-eight hours after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using M1-ubiquitin antibodies. The input was immunoblotted for NEMO and actin. PINK1 silencing efficiency was determined by real-time RT–PCR. Bars represent mean ± SD with three technical replicates. Source data are available online for this figure.

Fig 3: PINK1 stabilizes M1-linked ubiquitin by phosphorylation and counteracts OTULIN activity PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL-1L were transfected with PINK1, WT OTULIN, or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep-tagged UBAN domain of NEMO to enrich proteins modified with M1-linked ubiquitin. Proteins affinity-purified by Strep-Tactin beads were immunoblotted against ubiquitin.Catalytically active PINK1 increases p-S65-ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase-dead (K/D) PINK1. Forty-eight hours after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p-S65-ubiquitin, PINK1, and VDAC (loading control).The TNF-induced increase in p-S65-ubiquitin is abolished in PINK1-deficient cells. HEK293T cells were transfected with control or PINK1-specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies.TNF-induced M1-ubiquitination is reduced in PINK1-deficient cells. WT and PINK1-KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep-tagged UBAN domain, as described in (A), followed by immunoblotting using M1-ubiquitin-specific antibodies, Strep-Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1-ubiquitin-positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of three independent experiments; n.s., not significant, ***P < 0.001. A two-way ANOVA test was used to analyze statistical significance. Source data are available online for this figure.