rabbit polyclonal anti tim23  (Proteintech)

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a Western blots of OMV-stimulated HeLa cells show specific autophagosome/lysosome-dependent degradation of mitochondrial marker proteins (TOM20 and TIM23), but not of Golgi (GM130) or endoplasmic reticulum (PDI). Western blots are representative of 3 independent experiments. b DiO-labeled gonococcal OMVs from strain ATCC 49226 colocalize with MitoBright-labeled mitochondria in HeLa cells. Scale bar, 5 μm. c Live HeLa cell microscopy and 3D image reconstruction shows OMVs remain associated with Cascade Blue-labeled endosomes when delivered to MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. d Gonococcal OMVs from strain ATCC 49226 dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P < 10 −15 . e TEM of HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86 show mitochondrial disruption and capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, one-way ANOVA with post-hoc Tukey test for mitochondrial disruption (Mock-ATCC 49226 OMVs: P = 2 × 10 −11 ; Mock-ZJXSH86 OMVs: P = 2 × 10 −11 ), Kruskal–Wallis with posthoc Dunn test for mitochondria in mitophagy-like structures (Mock-ATCC 49226 OMVs: P = 1 × 10 −6 ; Mock-ZJXSH86 OMVs: P = 7 × 10 −6 ). f Quantitative real-time PCR showing a reduced mitochondrial to genomic DNA ratio in ATCC 49226 OMV-stimulated HeLa cells. Data are mean ± s.d.; n = 3 independent biological replicates, one-way ANOVA with post-hoc Tukey test. g Increased HSP60 and LAMP1 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P = 6 × 10 −9 . h Increased HSP60 and LC3 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test (LC3 puncta Mock-OMVs: P = 7 × 10 −12 ; LC3 colocalized HSP60 Mock-ZJXSH86 OMVs: P = 4 × 10 −15 ). Cells in d , e , g , h are from 3 independent experiments. Images in b , c are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Western Blot, Marker, Labeling, Microscopy, Fluorescence, Membrane, Two Tailed Test, Disruption, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a TEM of HeLa cells expressing gonococcal PorB from strain ATCC 49226 show mitochondrial capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, two-tailed Mann–Whitney test, P = 2 × 10 −10 . b Western blots showing degradation of mitochondrial proteins TOM20 and TIM23 in HeLa cells expressing gonococcal PorB from strains ATCC 49226 and ZJXSH86, but not for PorB from Neisseria mucosa . c Western blots showing that gonococcal OMVs expressing PorB from N. mucosa lost the ability to induce degradation of TOM20 and TIM23 in HeLa cells. d Gonococcal OMVs expressing PorB from N. mucosa lost the ability to dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 55 cells from 4 independent experiments, Kruskal–Wallis with posthoc Dunn test (Mock-N.g. PorB: P = 8 × 10 −13 ; N.g. PorB-N.m. PorB: P < 10 −15 ). e Flow cytom e try analysis of TMRM fluorescence intensity in HeLa cells stimulated with gonococcal OMVs expressing gonococcal PorB or PorB from N. mucosa . f Gonococcal PorB structure (pdb entry 4AUI) and sequence of PorB from strain ATCC 49226. Lysines are indicated in blue, or in magenta when located within the PorB channel and associated with ATP (orange) binding. g Western blots showing reduced degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q. h Reduced HSP60 and LC3 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 2 × 10 −9 ; WT PorB-PorB K117Q: P = 1 × 10 −5 ). i Reduced HSP60 and LAMP1 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 9 × 10 −8 ). j Western blots of HeLa 4KO cells showing p62 expression restores PorB K117Q-dependent degradation of TOM20 and TIM23, while expression of OPTN or NDP52 restores PorB K5-dependent degradation of TOM20 and TIM23. k Western blots showing impaired degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q, K5 or K null . l Western blots showing that knock-down of PINK1 in HeLa cells inhibits PorB K5-dependent degradation of TOM20 and TIM23, but not for PorB K117Q. Cells in a , h , i are from 3 independent experiments. Western blots in b , c , g , j , k , l are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Fluorescence, Sequencing, Binding Assay, Knockdown

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a Western blots showing that knock-down of p62 in HeLa cells inhibits PorB K117Q-dependent degradation of TOM20 and TIM23. b Reduced p62 and HSP60 colocalization in HeLa cells expressing PorB K5 or K null compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; WT PorB-PorB K5: P < 10 −15 ; WT PorB-PorB K null : P < 10 −15 ). c Western blots of HeLa 4KO cells expressing WT or truncated p62 show that the p62 LIR domain and UBI domain are indispensable for PorB K117Q-induced degradation of TOM20 and TIM23. d Western blots showing co-immunoprecipitation of ubiquitin and p62 after immunoprecipitation of PorB WT and PorB K117Q. e Western blots showing co-immunoprecipitation of both K63-linked and K48-linked polyubiquitin, after immunoprecipitation of PorB from HeLa cells, which is enhanced with inhibitors for autolysosomal degradation (BafA1, 3-MA) for K63-linked ubiquitin or proteasomal degradation (MG132) for K48-linked ubiquitin. f Colocalization between HSP60 and K63-linked polyubiquitin, but not K48-linked polyubiquitin, is induced in HeLa cells expressing PorB. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test, K63 only Empty-PorB K117Q: P < 10 −15 . g Western blots after immunoprecipitation of HA-PorB from HeLa cells show co-immunoprecipitation of K63-linked polyubiquitin is dependent on PorB lysine 171 and co-immunoprecipitation of K48-linked polyubiquitin is dependent on PorB lysine 128. h Western blots showing PorB-induced degradation of TOM20 and TIM23 in HeLa cells is dependent on PorB lysines 117 and 171. i Western blots showing gonococcal OMVs expressing PorB K117Q/K171Q lost the ability to induce degradation of TOM20 and TIM23. Cells in b and f are from 3 independent experiments. Western blots in a , c , d , e , g , h , i are representative of 3 independent exper i ments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Western Blot, Knockdown, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a MS/MS spectrum of RNF213 peptide identified by mass spectrometry analysis of proteins co-immunoprecipitated with PorB from HeLa cells. b Western blots after immunoprecipitation of PorB from HeLa cells show co-immunoprecipitation of RNF213. c HeLa cells expressing PorB show colocalization between PorB, HSP60 and RNF213. Scale bar, 5 μm. d Live HeLa cell microscopy and 3D image reconstruction shows RNF213 surrounding PorB on MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. e Western blots showing PorB K171-dependent enhanced co-immunoprecipitation of ubiquitin from HeLa cells transfected with an RNF213 expression vector. f Western blots showing that transfection of HeLa cells with RNF213 siRNA abolishes PorB K171-dependent co-immunoprecipitation of ubiquitin. g Western blots showing PorB K117Q, but not PorB K171Q, enhances PorB-induced degradation of TOM20 and TIM23 in HeLa cells transfected with an RNF213 expression vector. h Western blots showing that transfection of HeLa cells with RNF213 siRNA prevents PorB-induced degradation of TOM20 and TIM23 for PorB K117Q, while degradation remains unaffected for PorB K171Q. Western blots in b, e – h are representative of 3 independent experiments. Images in c and d are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Tandem Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation, Western Blot, Expressing, Microscopy, Labeling, Fluorescence, Ubiquitin Proteomics, Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: LPGAT1 controls MEGDEL syndrome by coupling phosphatidylglycerol remodeling with mitochondrial transport

doi: 10.1016/j.celrep.2023.113214

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-TIMM23 , Proteintech , Cat#11123–1-AP; RRID:AB_615045.

Techniques: Virus, Recombinant, Transfection, Acid Assay, Pyruvate Assay, Bioassay, H&E Stain, Staining, ROS Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Control, CRISPR, Software, Imaging