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 for VAP-B and myc-tag showing VAP-B expression in control and myc-VAP-B overexpressed U2OS cells. INF2 is used as loading control. Molecular weight in kDa. ( B ) Quantification of CCCP-induced actin polymerization in control and myc-VAP-B-overexpressing U2OS cells. Data with 65 cells (control) and 53 cells (overexpressing myc-VAP-B) from 3 independent runs. Error ± SEM. ( C ) Averaged traces showing mitochondrial calcium fold change (MitoGCamp6f) following histamine stimulation (at time 0) in control (ER-RFP expressing), ER-RFP-mito (ER-Mito linker) and PM-RFP-Mito (PM-Mito-linker) transfected HeLa cells. Data from 2 independent experiments comprising of 4 individual traces for each condition. Error: ±SEM. ( D ) Dot plot of peak fold change in mitochondrial calcium following histamine stimulation from individual traces as in Fig S4C. Data for 32 cells (Control- ER-RFP); 32 cells (ER-RFP-Mito); 28 cells (PM-RFP-Mito) for 3 independent runs. Error: ±s.d. P = 0.0213 (*) for Control Vs ER-RFP-Mito, P = 0.0439 (*) for Control Vs PM-RFP-Mito and P = 0.0001 (****) for ER-RFP-Mito Vs PM-RFP-Mito, One-way ANOVA used. ( E ) Graph showing actin filaments intensity (GFP-Ftractin) from HeLa cells transiently expressing either ER-RFP-Mito (magenta) or PM-RFP-Mito or ER-RFP, treated with 20 µM CCCP. Data from 28 cells (ER-RFP); 27 cells (ER-RFP-Mito) and 23 cells (PM-RFP-Mito) from 4 independent experiments. Error: ±SEM.
Article Snippet: Primary antibody: anti-myc-tag antibody (Abcam, ab32 used 1:1000) anti-INF2 antibody (CST #41081; used at 1:1000) anti-VAP-B antibody (Proteintech #14477-1-AP; used at 1:5000 at RT) anti-LC3B antibody (CST #12741S; used at 1:1000) anti-Parkin antibody (CST #2132S; used at 1:1000) anti-Parkin(phospho S65) antibody (Abcam #315376; used at 1:1000) anti-Pink1 antibody (CST #6946S; used at 1:1000) anti-β-actin antibody (CST #3700S; used at 1:10,000) anti-Mfn2 antibody (CST #9482S; used at 1:1000) anti-VDAC antibody (Invitrogen #MA5-41088; used at 1:1000) anti-Phospho ubiquitin (Ser65) (CST #62802S; used at 1:1000).
Techniques: Western Blot, Expressing, Control, Molecular Weight, Transfection