mouse anti-prkn  (Millipore)

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone reduces PRKN-mediated BAX degradation and promotes BAX-BAK1 mitochondrial pores formation. (A) WB assays were performed to assess the BAX and BAK1 proteins expression in Huh7 cells treated with ailanthone for 48 h. (B) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression in mitochondria and cytoplasm were measured by WB. (C, D) Huh7 cells were treated with 1.2 μM ailanthone for 48 h. The lysates were immunoprecipitated against BAX, and immunoblotting assays for BAX and BAK1 were performed (C) . The colocalization of BAX and BAK1 was observed (1000×, scale bar: 50 μm). The colocalization area of BAX with BAK1 per cell was quantified (D) . (E) Huh7 cells were treated with MSN-50 (1 μM for 3 h), MSN-50 was then removed, and the cells were treated with 0.6 μM ailanthone for 48 h. CCK8 assay measured cell viability. (F, G) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression were measured by WB (F) . The colocalization of BAX and PRKN proteins were observed (1000×, scale bar: 50 μm). The colocalization area of BAX with PRKN per cell was quantified (G) . (H, I) IP analysis of BAX and BAK1 in Huh7 cells treated with 1.2 μM ailanthone for 48 h was conducted. The ubiquitinated modified BAX and BAK1 signals were visualized using a pan-ubiquitin antibody. The quantitative analysis of ubiquitinated BAX and BAK1 proteins expression was normalized to their respective levels in the IP group. Bar, SD. * p < 0.05 or ** p < 0.01.

Article Snippet: The primary antibodies included antibodies against PRKN (CST, 4211, 1:250), BAK1 (CST, 12105, 1:250), BAX (Proteintech, 50599-2-Ig, 1:250).

Techniques: Expressing, Immunoprecipitation, Western Blot, CCK-8 Assay, Modification, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone blocks PINK1-PRKN-mediated mitophagy while promoting BAX-BAK1 mitochondrial pores formation and inducing inflammation in vivo . (A, B) IHC detection of LC3, PINK1, PRKN, BAX, BAK1, TNF-α, IL-1β, and IL-6 proteins expression in tumour tissues (100×, scale bar: 500 μm). (C, D) The colocalization of PINK1 with PRKN and BAX with BAK1 in tumour tissues was observed by confocal microscopy imaging (400×, scale bar: 100 μm). The colocalization areas were quantified. Bar, SD. ** p < 0.01.

Article Snippet: The primary antibodies included antibodies against PRKN (CST, 4211, 1:250), BAK1 (CST, 12105, 1:250), BAX (Proteintech, 50599-2-Ig, 1:250).

Techniques: In Vivo, Expressing, Confocal Microscopy, Imaging

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Schematic representation of ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation. Ailanthone induces mitochondrial damage while blocking PINK1-PRKN-mediated mitophagy, leading to the accumulation of dysfunctional mitochondria in cells, thereby inhibiting hepatocellular carcinoma (HCC) cell proliferation. Furthermore, the inhibition of PRKN protein by ailanthone reduced the ubiquitination and degradation of BAX, promoting the localization of BAX on the outer mitochondrial membrane, forming BAX-BAK1 mitochondrial pores, aggravating the release of mtDNA, and inducing inflammatory responses. Ultimately, the accumulation of damaged mitochondria and the release of inflammatory factors both contribute to the suppressive effect of ailanthone on HCC cell proliferation.

Article Snippet: The primary antibodies included antibodies against PRKN (CST, 4211, 1:250), BAK1 (CST, 12105, 1:250), BAX (Proteintech, 50599-2-Ig, 1:250).

Techniques: Blocking Assay, Inhibition, Ubiquitin Proteomics, Membrane

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone suppresses mitophagy by disturbing PINK1-PRKN axis. (A, B) WB assays were performed to assess the PINK1 and PRKN proteins expression in Huh7 cells treated with ailanthone (1.2 μM) for different times (A) , and treated with ailanthone for 48 h (B) . (C) Huh7 cells were treated by ailanthone for 48 h, PINK1 and PRKN mRNA levels were detected by qPCR assay. (D, E) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), the proteins expression of PINK1 and PRKN in whole cells (D) , and in mitochondria and cytoplasm (E) were measured by WB assay. (F) Immunoprecipitated against PRKN, and WB assays for PINK1 and PRKN were performed. Quantitative analysis of PINK1 expression normalised to that of PRKN in the IP group. (G) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h). The colocalization of PINK1 with PRKN was observed by confocal microscopy (1000×, scale bar: 50 μm). The colocalization area of PINK1 with PRKN per cell was quantified. Bar, SD . * p < 0.05 or ** p < 0.01.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: Expressing, Immunoprecipitation, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone reduces PRKN-mediated BAX degradation and promotes BAX-BAK1 mitochondrial pores formation. (A) WB assays were performed to assess the BAX and BAK1 proteins expression in Huh7 cells treated with ailanthone for 48 h. (B) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression in mitochondria and cytoplasm were measured by WB. (C, D) Huh7 cells were treated with 1.2 μM ailanthone for 48 h. The lysates were immunoprecipitated against BAX, and immunoblotting assays for BAX and BAK1 were performed (C) . The colocalization of BAX and BAK1 was observed (1000×, scale bar: 50 μm). The colocalization area of BAX with BAK1 per cell was quantified (D) . (E) Huh7 cells were treated with MSN-50 (1 μM for 3 h), MSN-50 was then removed, and the cells were treated with 0.6 μM ailanthone for 48 h. CCK8 assay measured cell viability. (F, G) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression were measured by WB (F) . The colocalization of BAX and PRKN proteins were observed (1000×, scale bar: 50 μm). The colocalization area of BAX with PRKN per cell was quantified (G) . (H, I) IP analysis of BAX and BAK1 in Huh7 cells treated with 1.2 μM ailanthone for 48 h was conducted. The ubiquitinated modified BAX and BAK1 signals were visualized using a pan-ubiquitin antibody. The quantitative analysis of ubiquitinated BAX and BAK1 proteins expression was normalized to their respective levels in the IP group. Bar, SD. * p < 0.05 or ** p < 0.01.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: Expressing, Immunoprecipitation, Western Blot, CCK-8 Assay, Modification, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone blocks PINK1-PRKN-mediated mitophagy while promoting BAX-BAK1 mitochondrial pores formation and inducing inflammation in vivo . (A, B) IHC detection of LC3, PINK1, PRKN, BAX, BAK1, TNF-α, IL-1β, and IL-6 proteins expression in tumour tissues (100×, scale bar: 500 μm). (C, D) The colocalization of PINK1 with PRKN and BAX with BAK1 in tumour tissues was observed by confocal microscopy imaging (400×, scale bar: 100 μm). The colocalization areas were quantified. Bar, SD. ** p < 0.01.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: In Vivo, Expressing, Confocal Microscopy, Imaging

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Schematic representation of ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation. Ailanthone induces mitochondrial damage while blocking PINK1-PRKN-mediated mitophagy, leading to the accumulation of dysfunctional mitochondria in cells, thereby inhibiting hepatocellular carcinoma (HCC) cell proliferation. Furthermore, the inhibition of PRKN protein by ailanthone reduced the ubiquitination and degradation of BAX, promoting the localization of BAX on the outer mitochondrial membrane, forming BAX-BAK1 mitochondrial pores, aggravating the release of mtDNA, and inducing inflammatory responses. Ultimately, the accumulation of damaged mitochondria and the release of inflammatory factors both contribute to the suppressive effect of ailanthone on HCC cell proliferation.

Article Snippet: Primary antibodies included those against ACTB/β-Actin (ABclonal Technology, AC026; 1:200000), LC3-I/II (CST, 12741 1:1000), COX4I1 (Proteintech, 66110-1-Ig, 1:8000), PINK1 (CST, 6946, 1:1000), PRKN (CST, 4211, 1:1000), BAK1 (Proteintech, 29552-1-AP, 1:10000), BAX (Proteintech, 50599-2-lg, 1:10000), Ub (CST, 3,936, 1:1000), TNF-α (Proteintech, 60291-1-Ig, 1:8000), IL-1β (Proteintech, 16806-1-AP, 1:1000), IL-6 (Proteintech, 21865-1-AP, 1:1000).

Techniques: Blocking Assay, Inhibition, Ubiquitin Proteomics, Membrane

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