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( A ) Western blot showing <t>Pink1</t> levels in HeLa cells overexpressing GFP-Pink1 treated with 20 µM CCCP for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( B ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of CCCP treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0145 (CCCP 20 Vs CK666 + CCCP 20) (*), P = 0.3731 (CCCP 40 vs CK666 + CCCP 40) (ns), one-way ANOVA used. ( C ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with a combination of 15 µM Oligomycin A and 25 µM Antimycin A for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( D ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of Oligomycin A + Antimycin A treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0017 (A/O 20 Vs CK666 + A/O 20) (**), P = 0.0291 (A/O 40 vs CK666 + A/O 40) (*), one-way ANOVA used. ( E ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with 20 µM CCCP in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. ( F ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with a combination of 15 µM Oligomycin A and 25 µM Antimycin A in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. .
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( A ) Western blot showing <t>Pink1</t> levels in HeLa cells overexpressing GFP-Pink1 treated with 20 µM CCCP for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( B ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of CCCP treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0145 (CCCP 20 Vs CK666 + CCCP 20) (*), P = 0.3731 (CCCP 40 vs CK666 + CCCP 40) (ns), one-way ANOVA used. ( C ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with a combination of 15 µM Oligomycin A and 25 µM Antimycin A for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( D ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of Oligomycin A + Antimycin A treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0017 (A/O 20 Vs CK666 + A/O 20) (**), P = 0.0291 (A/O 40 vs CK666 + A/O 40) (*), one-way ANOVA used. ( E ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with 20 µM CCCP in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. ( F ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with a combination of 15 µM Oligomycin A and 25 µM Antimycin A in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. .
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( A ) Western blot showing <t>Pink1</t> levels in HeLa cells overexpressing GFP-Pink1 treated with 20 µM CCCP for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( B ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of CCCP treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0145 (CCCP 20 Vs CK666 + CCCP 20) (*), P = 0.3731 (CCCP 40 vs CK666 + CCCP 40) (ns), one-way ANOVA used. ( C ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with a combination of 15 µM Oligomycin A and 25 µM Antimycin A for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( D ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of Oligomycin A + Antimycin A treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0017 (A/O 20 Vs CK666 + A/O 20) (**), P = 0.0291 (A/O 40 vs CK666 + A/O 40) (*), one-way ANOVA used. ( E ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with 20 µM CCCP in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. ( F ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with a combination of 15 µM Oligomycin A and 25 µM Antimycin A in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. .
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( A ) Western blot showing <t>Pink1</t> levels in HeLa cells overexpressing GFP-Pink1 treated with 20 µM CCCP for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( B ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of CCCP treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0145 (CCCP 20 Vs CK666 + CCCP 20) (*), P = 0.3731 (CCCP 40 vs CK666 + CCCP 40) (ns), one-way ANOVA used. ( C ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with a combination of 15 µM Oligomycin A and 25 µM Antimycin A for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( D ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of Oligomycin A + Antimycin A treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0017 (A/O 20 Vs CK666 + A/O 20) (**), P = 0.0291 (A/O 40 vs CK666 + A/O 40) (*), one-way ANOVA used. ( E ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with 20 µM CCCP in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. ( F ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with a combination of 15 µM Oligomycin A and 25 µM Antimycin A in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. .
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( A ) Western blot showing <t>Pink1</t> levels in HeLa cells overexpressing GFP-Pink1 treated with 20 µM CCCP for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( B ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of CCCP treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0145 (CCCP 20 Vs CK666 + CCCP 20) (*), P = 0.3731 (CCCP 40 vs CK666 + CCCP 40) (ns), one-way ANOVA used. ( C ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with a combination of 15 µM Oligomycin A and 25 µM Antimycin A for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( D ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of Oligomycin A + Antimycin A treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0017 (A/O 20 Vs CK666 + A/O 20) (**), P = 0.0291 (A/O 40 vs CK666 + A/O 40) (*), one-way ANOVA used. ( E ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with 20 µM CCCP in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. ( F ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with a combination of 15 µM Oligomycin A and 25 µM Antimycin A in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. .
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( A ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with 20 µM CCCP for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( B ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of CCCP treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0145 (CCCP 20 Vs CK666 + CCCP 20) (*), P = 0.3731 (CCCP 40 vs CK666 + CCCP 40) (ns), one-way ANOVA used. ( C ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with a combination of 15 µM Oligomycin A and 25 µM Antimycin A for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( D ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of Oligomycin A + Antimycin A treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0017 (A/O 20 Vs CK666 + A/O 20) (**), P = 0.0291 (A/O 40 vs CK666 + A/O 40) (*), one-way ANOVA used. ( E ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with 20 µM CCCP in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. ( F ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with a combination of 15 µM Oligomycin A and 25 µM Antimycin A in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. .

Journal: EMBO Reports

Article Title: Peri-mitochondrial actin filaments inhibit Parkin assembly by disrupting ER-mitochondria contacts

doi: 10.1038/s44319-025-00561-y

Figure Lengend Snippet: ( A ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with 20 µM CCCP for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( B ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of CCCP treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0145 (CCCP 20 Vs CK666 + CCCP 20) (*), P = 0.3731 (CCCP 40 vs CK666 + CCCP 40) (ns), one-way ANOVA used. ( C ) Western blot showing Pink1 levels in HeLa cells overexpressing GFP-Pink1 treated with a combination of 15 µM Oligomycin A and 25 µM Antimycin A for 20 and 40 min in the presence or absence of 100 µM CK666. Actin used as loading control. Molecular weight in kDa. ( D ) Quantification showing relative Pink1 levels in HeLa cells overexpressing GFP-Pink1 following 20 and 40 min of Oligomycin A + Antimycin A treatment in the presence or absence of CK666 (100 µM). N = 3 independent experiments; Error: Mean ±s.d.; P = 0.0017 (A/O 20 Vs CK666 + A/O 20) (**), P = 0.0291 (A/O 40 vs CK666 + A/O 40) (*), one-way ANOVA used. ( E ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with 20 µM CCCP in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. ( F ) Western blot of HeLa cells overexpressing Parkin GFP showing Parkin, phospho-Parkin (S65) and phospho-ubiquitin (S65) upon treatment with a combination of 15 µM Oligomycin A and 25 µM Antimycin A in the presence or absence of CK666 (100 µM) for various time points as shown. Molecular weight in kDa. .

Article Snippet: pcDNA-DEST47 PINK1 C-GFP , Mark Cookson , Addgene: #13316.

Techniques: Western Blot, Control, Molecular Weight, Ubiquitin Proteomics