rabbit polyclonal anti v5 tag antibody  (Biorbyt)

Journal: bioRxiv

Article Title: Lysosomal uptake of mtDNA mitigates heteroplasmy

doi: 10.1101/2024.02.16.580263

Figure Lengend Snippet: mtDNA damage follows a degradation pathway related to lysosomes. ( A ) Graphical model for the proximity proteomics approach. TurboID was split and combined with the mitochondrial outer membrane protein SAMM50 and the early endosomal marker RAB5C ( biorender.com ) ( B ) Volcano Plot showing proteins enriched after biotinylation and purification of cells expressing SplitTurboID plasmids and K319E-Cherry. Differentially expressed proteins compared with cells transduced with SAMM50-SplitCt (significant: q-value < -0.05 and absolute log2 fold change>1) are highlighted in blue (n=6). ( C ) Pathway enrichment analysis with Metascape showing GO terms for proteins differentially enriched. ( D ) Immunostaining and ( E ) quantification of HeLa cells expressing RAB10-GFP, RAB10 Q68L -GFP and TWNK K319E -mCherry plasmids and labeled with α-VPS35 and α-RAB5, and, ( F ) cells labeled with α-dsDNA and the lysosomal marker α-LAMP1 (n=3, 10 images per replicate). ( G ) Quantification of cytosolic dsDNA foci in cells expressing RAB10-GFP, RAB10 Q68L -GFP and TWNK K319E -Cherry plasmids (n=3, <15 cells per replicate). ( H ) Manderścorrelation coefficient between RAB10-GFP and LAMP1, and ( I ) LAMP1 and dsDNA (n=3, 10 images per replicate). j ) RAB10-GFP co-Immunoprecipitation in steady state and ( K ) in cells expressing TWNK K319E -Cherry with the lysosomal protein LAMP1. P values were calculated using One-way ANOVA with Tukey correction for multiple comparison. Scale bar, 10 μm. Data is presented as mean ± SEM

Article Snippet: Antibodies used for western blot were: polyclonal α-SDHB (10620-1-AP; 1:1000), α-optineurin (10837-1-AP; 1:1000) α-LC3B (14600-1-AP; 1:1000), α-GFP (50430-2-AP; 1:1000) and monoclonal α-GAPDH (60004-1-Ig; 1:4000), α-GFP (66002-1-Ig; 1:1000) and α-p62 (66184-1-Ig; 1:1000) from Proteintech; polyclonal α-SAMM50 (ab133709; 1:1000), α-RAB5 (ab109534; 1:1000) and monoclonal α-ATP5A (ab14748; 1:1000) from Abcam; polyclonal α-LAMP1 (#9091; 1:1000), α-V5 (#13202; 1:1000) and monoclonal α-V5 (#80076; 1:1000) from Cell Signaling; monoclonal α-VPS35 (sc-374372; 1:1000) and α-RAB5 (sc-46692; 1:500) from Santa Cruz.

Techniques: Membrane, Marker, Purification, Expressing, Transduction, Immunostaining, Labeling, Immunoprecipitation, Comparison

Journal: bioRxiv

Article Title: Lysosomal uptake of mtDNA mitigates heteroplasmy

doi: 10.1101/2024.02.16.580263

Figure Lengend Snippet: ( A ) Immunostaining of RAB10-GFP and RAB10 Q68L -GFP transfected cells labelled with α-VPS35, the endoplasmic reticulum (ER) marker α-Calnexin (CNX) and ( B ) α-LAMP1 and α-dsDNA. ( C ) Manderścorrelation coefficient between RAB10 and Calnexin. (n=3, 10 images per replicate). ( D ) co-Immunoprecipitation of α-RAB5 with RAB10 and LAMP1. ( E, F ) Immunostaining and fluorescence profile of cells transfected with VPS35-GFP and VPS35 D620N -GFP and labelled with α-RAB5 and α-PDH. ( G ) Cells transfected with VPS35-GFP plasmids and stained with mitochondrial markers α-TOM20, α-PDH and α-dsDNA. Scale bar, 10 μm. Data is presented as mean ± SEM.

Article Snippet: Antibodies used for western blot were: polyclonal α-SDHB (10620-1-AP; 1:1000), α-optineurin (10837-1-AP; 1:1000) α-LC3B (14600-1-AP; 1:1000), α-GFP (50430-2-AP; 1:1000) and monoclonal α-GAPDH (60004-1-Ig; 1:4000), α-GFP (66002-1-Ig; 1:1000) and α-p62 (66184-1-Ig; 1:1000) from Proteintech; polyclonal α-SAMM50 (ab133709; 1:1000), α-RAB5 (ab109534; 1:1000) and monoclonal α-ATP5A (ab14748; 1:1000) from Abcam; polyclonal α-LAMP1 (#9091; 1:1000), α-V5 (#13202; 1:1000) and monoclonal α-V5 (#80076; 1:1000) from Cell Signaling; monoclonal α-VPS35 (sc-374372; 1:1000) and α-RAB5 (sc-46692; 1:500) from Santa Cruz.

Techniques: Immunostaining, Transfection, Marker, Immunoprecipitation, Fluorescence, Staining

Journal: bioRxiv

Article Title: Lysosomal uptake of mtDNA mitigates heteroplasmy

doi: 10.1101/2024.02.16.580263

Figure Lengend Snippet: VPS35 overexpression accelerates autophagy flux through lysosomal accumulation. ( A ) Western blot analysis and ( B ) quantification of macroautophagy and proteins from the vesicular system in control and cell expressing VPS35-V5 (n=3). ( C ) Western blot and ( D ) quantification in cells treated with 10 μM Chloroquine (Cq) for 4h to assay autophagy flux (n=4). ( E ) Western blot and ( F ) quantification in SAMM50 knock-down cells overexpressing VPS35-V5. α-GAPDH was used as a loading control (n=4). ( G ) mtDNA copy number quantification for control and VPS35 cells in steady state and ( H ) upon SAMM50 KD (+ Dox) (n=4). ( I ) α-ATP5A and α-LAMP1 immunostaining in basal and upon SAMM50 KD (+ Dox) in VPS35-V5 cells. Arrows depict colocalization foci. ( J ) Manderś correlation coefficient between LAMP1 and ATP5A and vice versa (n=3, 10 images per replicate) ( K ) α-dsDNA and α-TOM20 immunostaining in VPS35-V5 and SAMM50 KD cells. ( L ) mRNA quantification of innate immune related genes. GAPDH mRNA was used for normalization (n=4). P values were calculated using Studentś T-test (B, L) and One-way ANOVA with Tukey correction for multiple comparison (J). Scale bar, 10 μm. Data is presented as mean ± SEM.

Article Snippet: Antibodies used for western blot were: polyclonal α-SDHB (10620-1-AP; 1:1000), α-optineurin (10837-1-AP; 1:1000) α-LC3B (14600-1-AP; 1:1000), α-GFP (50430-2-AP; 1:1000) and monoclonal α-GAPDH (60004-1-Ig; 1:4000), α-GFP (66002-1-Ig; 1:1000) and α-p62 (66184-1-Ig; 1:1000) from Proteintech; polyclonal α-SAMM50 (ab133709; 1:1000), α-RAB5 (ab109534; 1:1000) and monoclonal α-ATP5A (ab14748; 1:1000) from Abcam; polyclonal α-LAMP1 (#9091; 1:1000), α-V5 (#13202; 1:1000) and monoclonal α-V5 (#80076; 1:1000) from Cell Signaling; monoclonal α-VPS35 (sc-374372; 1:1000) and α-RAB5 (sc-46692; 1:500) from Santa Cruz.

Techniques: Over Expression, Western Blot, Control, Expressing, Knockdown, Immunostaining, Comparison