Journal: Autophagy

Article Title: Basal activity of PINK1 and PRKN in cell models and rodent brain

doi: 10.1080/15548627.2023.2286414

Figure Lengend Snippet: Complete loss of PINK1 in mouse brain stabilizes PRKN protein while partial loss dampens p-S65-Ub levels. PRKN and p-S65-Ub levels were determined at basal conditions in hemibrain lysates from mice with the following genotypes: WT ( n = 29), heterozygous Prkn +/- ( n = 23), homozygous prkn -/- ( n = 29), heterozygous Pink1 +/- ( n = 15), homozygous pink1 -/- ( n = 22). For better comparison, mice were grouped and analyzed by genotype: WT, prkn -/- , pink1 -/- (left); WT, Prkn +/- , prkn -/- (middle); and WT, Pink1 +/- , pink1 -/- (right). (A) Representative western blots obtained with three different anti-PRKN antibodies (Prk8, 2132, and 5C3) are shown together with the loading control GAPDH. An open arrowhead labels the truncated PRKN-EGFP fusion protein produced in the prkn -/- samples. (B) PRKN (Prk8) protein levels were assessed by densitometry with data points shown as ratio PRKN divided by GAPDH (median ± interquartile range [IQR]). Quantification of PRKN protein using the other two antibodies can be found in Fig. S1A and B. (C) p-S65-Ub levels were quantified by sandwich ELISA with data points shown as median ± IQR. Data was analyzed by using a Kruskal-Wallis test combined with Dunn’s multiple comparison test (***, p < 0.0005; *, p < 0.05). Comparisons to the WT are shown on top of data points, while comparisons between other genotypes are indicated by brackets.

Article Snippet: Membranes were blocked with 5% skim milk in TBS with 0.1% Tween (TBST) and incubated overnight with the following primary antibodies: rabbit anti-PINK1 (Cell Signaling Technology, 6946; 1:2000), mouse anti-PINK1 (Biolegend, 846201; 1:1000), mouse anti-PRKN (Millipore, MAB5521; Prk8; 1:1000–5000 for cell lysates), mouse anti-PRKN (Cell Signaling Technology, 4211; Prk8; 1:25,000 for rodent lysates]), mouse anti-PRKN (Biolegend, 865602; 5C3; 1:2,000 for rodent lysates), rabbit anti-PRKN (Cell Signaling Technology, 2132; 1:2,000 for rodent lysates), rabbit anti-p-S65-Ub (Cell Signaling Technology, 62802; 1:3000–20,000), rabbit anti-GFP (Takara/Clontech, 632460; 1:1,000 for rodent lysates) mouse anti-MFN2 (Abcam, ab56889; 1:2000), rabbit anti-TUBB3/betaIII-tubulin (Cell Signaling Technology, 5568; 1:10,000), rabbit anti-TH/tyrosine hydroxylase (Millipore, ab152, 1:1000), mouse anti-GAPDH (Meridian Life science, H86504M: 1:400,000), mouse anti-VCL/vinculin (Sigma-Aldrich, V9131; 1:500,000–1,500,000).

Techniques: Comparison, Western Blot, Produced, Sandwich ELISA

Journal: Autophagy

Article Title: Basal activity of PINK1 and PRKN in cell models and rodent brain

doi: 10.1080/15548627.2023.2286414

Figure Lengend Snippet: PINK1 gene dosage affects p-S65-Ub and PRKN levels in PD patients’ cells. Primary skin fibroblasts from related individuals without (QQ; n = 2), with one (QX; n = 3) or with two (XX; n = 2) mutant PINK1 Q456X alleles were either left untreated (0 h, left) or were treated for 2 h (middle) or 8 h (right) with 1 µM valinomycin (Val). Data is arranged by treatment groups (i.e., time points). (A) Cell lysates were analyzed by western blot using antibodies against PINK1, PRKN, and the loading control VCL (vinculin). (B) Protein levels of PINK1 were quantified by sandwich ELISA. (C) PRKN protein levels were derived by densitometry of the western blots and data points shown as a ratio of PRKN divided by VCL and normalized to PINK1 XX . (D) p-S65-Ub levels were also quantified by sandwich ELISA. Data is shown as the mean ± standard error of the mean of biological replicates, grouped by allele count of the Q456X mutation, and analyzed by one-way ANOVA with Tukey’s multiple comparison test (***, p < 0.0005; **, p < 0.005; *, p < 0.05). For untreated the mean of 4 technical repeats per biological sample is shown. Asterisks on top of data points indicate individual comparison to WT controls without PINK1 mutation.

Article Snippet: Membranes were blocked with 5% skim milk in TBS with 0.1% Tween (TBST) and incubated overnight with the following primary antibodies: rabbit anti-PINK1 (Cell Signaling Technology, 6946; 1:2000), mouse anti-PINK1 (Biolegend, 846201; 1:1000), mouse anti-PRKN (Millipore, MAB5521; Prk8; 1:1000–5000 for cell lysates), mouse anti-PRKN (Cell Signaling Technology, 4211; Prk8; 1:25,000 for rodent lysates]), mouse anti-PRKN (Biolegend, 865602; 5C3; 1:2,000 for rodent lysates), rabbit anti-PRKN (Cell Signaling Technology, 2132; 1:2,000 for rodent lysates), rabbit anti-p-S65-Ub (Cell Signaling Technology, 62802; 1:3000–20,000), rabbit anti-GFP (Takara/Clontech, 632460; 1:1,000 for rodent lysates) mouse anti-MFN2 (Abcam, ab56889; 1:2000), rabbit anti-TUBB3/betaIII-tubulin (Cell Signaling Technology, 5568; 1:10,000), rabbit anti-TH/tyrosine hydroxylase (Millipore, ab152, 1:1000), mouse anti-GAPDH (Meridian Life science, H86504M: 1:400,000), mouse anti-VCL/vinculin (Sigma-Aldrich, V9131; 1:500,000–1,500,000).

Techniques: Mutagenesis, Western Blot, Sandwich ELISA, Derivative Assay, Comparison

Journal: Autophagy

Article Title: Basal activity of PINK1 and PRKN in cell models and rodent brain

doi: 10.1080/15548627.2023.2286414

Figure Lengend Snippet: Cell type-specific expression of PINK1 and PRKN in fibroblasts, iPSCs, and midbrain DA neurons drive p-S65-Ub levels. Primary skin fibroblasts from either a control or patient with PINK1 I368N mutation, and undifferentiated iPS cells that were generated from the same PINK1 I368N patient cells and their gene-corrected counterparts (isogenic WT), as well as DA neurons generated from the same iPSC set were treated with 1 µM valinomycin (Val) for the indicated times and harvested. (A) Representative western blots show levels of PINK1, PRKN, p-S65-Ub and MFN2 for all three cell lines. VCL (vinculin) and DA neuronal markers, TUBB3/βIII-tubulin and TH (tyrosine hydroxylase), were used as loading controls. (B) Quantification of PINK1 protein levels by sandwich ELISA. (C) Densitometric analysis of PRKN western blots shown under (A) and data points displayed as PRKN divided by VCL or PRKN divided by DA neuronal markers. (D) Quantification of p-S65-Ub levels measured by sandwich ELISA. (E) Densitometric analysis of MFN2. Relative modification of MFN2 was calculated as the ratio of upper (ubiquitinated) to lower (unmodified) MFN2 band. The mean of three independent experiments for each cell type ± SD is shown. Statistical analysis was performed by one-way ANOVA followed by Bonferroni correction. (***, p < 0.0005; **, p < 0.005; *, p < 0.05). Asterisks on top of data points indicate individual comparison to respective WT controls without PINK1 mutation.

Article Snippet: Membranes were blocked with 5% skim milk in TBS with 0.1% Tween (TBST) and incubated overnight with the following primary antibodies: rabbit anti-PINK1 (Cell Signaling Technology, 6946; 1:2000), mouse anti-PINK1 (Biolegend, 846201; 1:1000), mouse anti-PRKN (Millipore, MAB5521; Prk8; 1:1000–5000 for cell lysates), mouse anti-PRKN (Cell Signaling Technology, 4211; Prk8; 1:25,000 for rodent lysates]), mouse anti-PRKN (Biolegend, 865602; 5C3; 1:2,000 for rodent lysates), rabbit anti-PRKN (Cell Signaling Technology, 2132; 1:2,000 for rodent lysates), rabbit anti-p-S65-Ub (Cell Signaling Technology, 62802; 1:3000–20,000), rabbit anti-GFP (Takara/Clontech, 632460; 1:1,000 for rodent lysates) mouse anti-MFN2 (Abcam, ab56889; 1:2000), rabbit anti-TUBB3/betaIII-tubulin (Cell Signaling Technology, 5568; 1:10,000), rabbit anti-TH/tyrosine hydroxylase (Millipore, ab152, 1:1000), mouse anti-GAPDH (Meridian Life science, H86504M: 1:400,000), mouse anti-VCL/vinculin (Sigma-Aldrich, V9131; 1:500,000–1,500,000).

Techniques: Expressing, Mutagenesis, Generated, Western Blot, Sandwich ELISA, Modification, Comparison

Journal: Molecular and Cellular Biology

Article Title: HSPA1A-Independent Suppression of PARK2 C289G Protein Aggregation by Human Small Heat Shock Proteins

doi: 10.1128/MCB.00698-14

Figure Lengend Snippet: Suppression of PARK2 C289G aggregation by HSPBs. (A) HEK293 cells were transfected for 24 h with Flag-tagged PARK2 WT or PARK2 C289G. Subcellular distribution of PARK2 (green) was investigated by immunofluorescence. The diagram shows the percentage of PARK2 aggregates. (B) Cells, transfected as in panel A, were fractionated in TX-100-soluble and -insoluble proteins and analyzed by Western blotting. (C) HEK293 cells were transfected for 24 h with Flag-tagged PARK2 C289G alone or together with each of the different HSPB proteins. Cells were fractionated in TX-100-soluble and -insoluble proteins and analyzed by Western blotting. The HSPB expression levels are shown in Fig. S1A in the supplemental material. (D) Immunofluorescent staining of Flag-tagged PARK2 (green), HSPBs (red), and DAPI (blue) in HEK293 cells, transfected as in panel C. (E) Diagram showing the percentages of flag-tagged PARK2-expressing cells with aggregates (*, P < 0.05; **, P < 0.001; n > three independent samples, means ± the SEM). Cells were divided in different categories, as shown in Fig. S1B in the supplemental material. HSPB6 was added as a negative control. HMW, high molecular weight.

Article Snippet: Mouse monoclonal anti-PARK2 antibody was obtained from Cell Signaling Technology.

Techniques: Transfection, Immunofluorescence, Western Blot, Expressing, Staining, Negative Control, High Molecular Weight

Journal: Molecular and Cellular Biology

Article Title: HSPA1A-Independent Suppression of PARK2 C289G Protein Aggregation by Human Small Heat Shock Proteins

doi: 10.1128/MCB.00698-14

Figure Lengend Snippet: Colocalization of the HSPBs with PARK2 C289G aggregates. (A) Immunofluorescent staining of Flag-tagged PARK2 (green), HSPBs (red), and DAPI (blue) in HEK293 cells, transfected as for Fig. 1D.

Article Snippet: Mouse monoclonal anti-PARK2 antibody was obtained from Cell Signaling Technology.

Techniques: Staining, Transfection

Journal: Molecular and Cellular Biology

Article Title: HSPA1A-Independent Suppression of PARK2 C289G Protein Aggregation by Human Small Heat Shock Proteins

doi: 10.1128/MCB.00698-14

Figure Lengend Snippet: HSPB1, HSPB2, HSPB4, and HSPB7 suppress PARK2 C289G aggregation in an HSPA1A-independent manner. (A) HEK293 cells were transfected with nonsense or HSPA1A RNAi. At 48 h posttransfection, the cells were transfected with Flag-tagged PARK2 C289G and mRFP-, HSPB1-, HSPB2-, HSPB4-, or HSPB7-encoding vectors and fractionated 24 h later in TX-100-soluble and -insoluble proteins. HMW, high molecular weight. (B) HEK293 cells were transfected for 24 h with Flag-tagged PARK2 C289G and mRFP-, HSPB1-, HSPB2-, HSPB4-, or HSPB7-encoding vector. Cells were treated with VER-155008 (40 μM, 24 h) and fractionated in TX-100-soluble and -insoluble proteins.

Article Snippet: Mouse monoclonal anti-PARK2 antibody was obtained from Cell Signaling Technology.

Techniques: Transfection, High Molecular Weight, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: HSPA1A-Independent Suppression of PARK2 C289G Protein Aggregation by Human Small Heat Shock Proteins

doi: 10.1128/MCB.00698-14

Figure Lengend Snippet: Role of ubiquitin proteasome system or the autophagy pathway in HSPB effects on PARK2 C289G aggregates. (A) HEK293 cells, transfected as in Fig. 3A, were treated with bortezomib (100 nM) or with 3-MA (20 mM) and wortmannin (200 nM) overnight. Cells were fractionated in TX-100 and analyzed for PARK2 C298G aggregates (*, P < 0.05; **, P < 0.001; n > three independent samples, means ± the SEM). (B) ATG5−/− MEF cells were transfected for 24 h with Flag-tagged PARK2 C289G and mRFP-, HSPB1-, HSPB2-, HSPB4-, or HSPB7-encoding vector and analyzed for PARK2 C298G aggregates (*, P < 0.05; **, P < 0.001; n > three independent samples, means ± the SEM). HMW, high molecular weight.

Article Snippet: Mouse monoclonal anti-PARK2 antibody was obtained from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, High Molecular Weight