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rabbit anti alg2  (Proteintech)


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    Structured Review

    Proteintech rabbit anti alg2
    Rabbit Anti Alg2, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/alg2/PDCD6+Antibody/pmc12865045-37-0-3
    Average 94 stars, based on 39 article reviews
    rabbit anti alg2 - by Bioz Stars, 2026-11
    94/100 stars

    Images

    Related Articles

    other:

    Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair.
    Article Snippet: IST1 (51002-1-AP, IF 1:200), CHMP2A (10477-1-AP, IF 1:100) and ALG-2 (12303-1-AP, IF 1:50) were purchased from Proteintech.

    Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair
    Article Snippet: Rabbit anti-ALG2 , Proteintech , Cat# 12303-1-AP.

    Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair
    Article Snippet: IST1 (51002-1-AP, IF 1:200), CHMP2A (10477-1-AP, IF 1:100) and ALG-2 (12303-1-AP, IF 1:50) were purchased from Proteintech.

    Transmission Assay:

    Article Title: Characteristics of extracellular vesicle-derived lncRNAs during the progression of HBV-related hepatocellular carcinoma
    Article Snippet: The particle size distribution of EVs was detected by nano-flow cytometry (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China). .. The morphology of EVs was observed by transmission electron microscopy (Transmission Electron Microscope H-7650, Hitachi High-Tech Corporation, Tokyo, Japan ) with uranyl acetate staining, and the marker proteins tumor Susceptibility 101(TSG101, ab125011, Abcam, Cambridge, UK), ALG-2-interacting protein 1(Alix, ab186429, Abcam, Cambridge, UK), CD9 (ab263019, Abcam, Cambridge, UK), and negative control Calnexin (10427-2-AP, Proteintech Group, Inc., Rosemont, IL, USA) were detected by Western Blot [ ]. .. Total RNA was isolated from EVs using the RNA Purification Kit (Simgen, cat. 5202050).

    Electron Microscopy:

    Article Title: Characteristics of extracellular vesicle-derived lncRNAs during the progression of HBV-related hepatocellular carcinoma
    Article Snippet: The particle size distribution of EVs was detected by nano-flow cytometry (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China). .. The morphology of EVs was observed by transmission electron microscopy (Transmission Electron Microscope H-7650, Hitachi High-Tech Corporation, Tokyo, Japan ) with uranyl acetate staining, and the marker proteins tumor Susceptibility 101(TSG101, ab125011, Abcam, Cambridge, UK), ALG-2-interacting protein 1(Alix, ab186429, Abcam, Cambridge, UK), CD9 (ab263019, Abcam, Cambridge, UK), and negative control Calnexin (10427-2-AP, Proteintech Group, Inc., Rosemont, IL, USA) were detected by Western Blot [ ]. .. Total RNA was isolated from EVs using the RNA Purification Kit (Simgen, cat. 5202050).

    Microscopy:

    Article Title: Characteristics of extracellular vesicle-derived lncRNAs during the progression of HBV-related hepatocellular carcinoma
    Article Snippet: The particle size distribution of EVs was detected by nano-flow cytometry (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China). .. The morphology of EVs was observed by transmission electron microscopy (Transmission Electron Microscope H-7650, Hitachi High-Tech Corporation, Tokyo, Japan ) with uranyl acetate staining, and the marker proteins tumor Susceptibility 101(TSG101, ab125011, Abcam, Cambridge, UK), ALG-2-interacting protein 1(Alix, ab186429, Abcam, Cambridge, UK), CD9 (ab263019, Abcam, Cambridge, UK), and negative control Calnexin (10427-2-AP, Proteintech Group, Inc., Rosemont, IL, USA) were detected by Western Blot [ ]. .. Total RNA was isolated from EVs using the RNA Purification Kit (Simgen, cat. 5202050).

    Staining:

    Article Title: Characteristics of extracellular vesicle-derived lncRNAs during the progression of HBV-related hepatocellular carcinoma
    Article Snippet: The particle size distribution of EVs was detected by nano-flow cytometry (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China). .. The morphology of EVs was observed by transmission electron microscopy (Transmission Electron Microscope H-7650, Hitachi High-Tech Corporation, Tokyo, Japan ) with uranyl acetate staining, and the marker proteins tumor Susceptibility 101(TSG101, ab125011, Abcam, Cambridge, UK), ALG-2-interacting protein 1(Alix, ab186429, Abcam, Cambridge, UK), CD9 (ab263019, Abcam, Cambridge, UK), and negative control Calnexin (10427-2-AP, Proteintech Group, Inc., Rosemont, IL, USA) were detected by Western Blot [ ]. .. Total RNA was isolated from EVs using the RNA Purification Kit (Simgen, cat. 5202050).

    Marker:

    Article Title: Characteristics of extracellular vesicle-derived lncRNAs during the progression of HBV-related hepatocellular carcinoma
    Article Snippet: The particle size distribution of EVs was detected by nano-flow cytometry (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China). .. The morphology of EVs was observed by transmission electron microscopy (Transmission Electron Microscope H-7650, Hitachi High-Tech Corporation, Tokyo, Japan ) with uranyl acetate staining, and the marker proteins tumor Susceptibility 101(TSG101, ab125011, Abcam, Cambridge, UK), ALG-2-interacting protein 1(Alix, ab186429, Abcam, Cambridge, UK), CD9 (ab263019, Abcam, Cambridge, UK), and negative control Calnexin (10427-2-AP, Proteintech Group, Inc., Rosemont, IL, USA) were detected by Western Blot [ ]. .. Total RNA was isolated from EVs using the RNA Purification Kit (Simgen, cat. 5202050).

    Negative Control:

    Article Title: Characteristics of extracellular vesicle-derived lncRNAs during the progression of HBV-related hepatocellular carcinoma
    Article Snippet: The particle size distribution of EVs was detected by nano-flow cytometry (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China). .. The morphology of EVs was observed by transmission electron microscopy (Transmission Electron Microscope H-7650, Hitachi High-Tech Corporation, Tokyo, Japan ) with uranyl acetate staining, and the marker proteins tumor Susceptibility 101(TSG101, ab125011, Abcam, Cambridge, UK), ALG-2-interacting protein 1(Alix, ab186429, Abcam, Cambridge, UK), CD9 (ab263019, Abcam, Cambridge, UK), and negative control Calnexin (10427-2-AP, Proteintech Group, Inc., Rosemont, IL, USA) were detected by Western Blot [ ]. .. Total RNA was isolated from EVs using the RNA Purification Kit (Simgen, cat. 5202050).

    Western Blot:

    Article Title: Characteristics of extracellular vesicle-derived lncRNAs during the progression of HBV-related hepatocellular carcinoma
    Article Snippet: The particle size distribution of EVs was detected by nano-flow cytometry (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China). .. The morphology of EVs was observed by transmission electron microscopy (Transmission Electron Microscope H-7650, Hitachi High-Tech Corporation, Tokyo, Japan ) with uranyl acetate staining, and the marker proteins tumor Susceptibility 101(TSG101, ab125011, Abcam, Cambridge, UK), ALG-2-interacting protein 1(Alix, ab186429, Abcam, Cambridge, UK), CD9 (ab263019, Abcam, Cambridge, UK), and negative control Calnexin (10427-2-AP, Proteintech Group, Inc., Rosemont, IL, USA) were detected by Western Blot [ ]. .. Total RNA was isolated from EVs using the RNA Purification Kit (Simgen, cat. 5202050).

    Article Title: Exosomes derived from human umbilical cord mesenchymal stem cells attenuate hepatic ischaemia-reperfusion injury via the let-7i-5p/Faslg axis
    Article Snippet: Additionally, fluorescence labelling of CD9 and CD81 (BD, Bergen, NJ, United States) was performed. .. Western blotting was performed to detect the markers ALG-2-interacting protein X (Alix), tumour susceptibility 101 (Tsg101), CD9, and CD63 (all antibodies were from Proteintech, Wuhan, China) in hucMSC-exos. ..

    Membrane:

    Article Title: tsRNA-12391-Modified Adipose Mesenchymal Stem Cell-Derived Exosomes Mitigate Cartilage Degeneration in Osteoarthritis by Enhancing Mitophagy.
    Article Snippet: Osteoarthritis (OA) is a cartilage-degenerative joint disease.. Mitophagy impacts articular cartilage damage. tRNA-derived small RNAs (tsRNAs) are one of the contents of adipose mesenchymal stem cell (AMSC)-derived exosomes (AMSC-exos) and are involved in disease progression.. However, whether tsRNAs regulatemitophagy andwhether tsRNA-modifiedAMSC-exos improve OA via mitophagy remain unclear.

    Incubation:

    Article Title: tsRNA-12391-Modified Adipose Mesenchymal Stem Cell-Derived Exosomes Mitigate Cartilage Degeneration in Osteoarthritis by Enhancing Mitophagy.
    Article Snippet: Osteoarthritis (OA) is a cartilage-degenerative joint disease.. Mitophagy impacts articular cartilage damage. tRNA-derived small RNAs (tsRNAs) are one of the contents of adipose mesenchymal stem cell (AMSC)-derived exosomes (AMSC-exos) and are involved in disease progression.. However, whether tsRNAs regulatemitophagy andwhether tsRNA-modifiedAMSC-exos improve OA via mitophagy remain unclear.



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    Novus Biologicals recombinant human alg2 protein
    ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or <t>ALG2</t> siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .
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    Proteintech rabbit alg2
    ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or <t>ALG2</t> siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .
    Rabbit Alg2, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

    Article Snippet: Recombinant Human ALIX protein (ab132534) from Abcam; Recombinant Human ALG2 Protein (H00085365-P01), Recombinant GST Epitope Tag Protein (NBC1-18537), Recombinant Human PACT His Protein (NBP2-51787) from NOVUS; Recombinant Human PKR Protein (LS-G22902-20) from LSBio.

    Techniques: Transfection, Control, Knockdown, Western Blot, Phospho-proteomics

    ( A ) Quantification by HCM of LAMP2 in U2OS cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). White masks, algorithm-defined cell boundaries; green masks, computer-identified LAMP2 puncta. ( B ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ), subjected to 2 mM LLOMe treatment for 30 min. ( D ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ), or pre-treated with 15 µM BAPTA-AM for 1 h. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALG2 knockdown cells (ALG2 KD ) overexpressing FLAG or FLAG-ALG2. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) Quantification by HCM of LAMP2 in U2OS cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). White masks, algorithm-defined cell boundaries; green masks, computer-identified LAMP2 puncta. ( B ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ), subjected to 2 mM LLOMe treatment for 30 min. ( D ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ), or pre-treated with 15 µM BAPTA-AM for 1 h. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALG2 knockdown cells (ALG2 KD ) overexpressing FLAG or FLAG-ALG2. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. .

    Article Snippet: Recombinant Human ALIX protein (ab132534) from Abcam; Recombinant Human ALG2 Protein (H00085365-P01), Recombinant GST Epitope Tag Protein (NBC1-18537), Recombinant Human PACT His Protein (NBP2-51787) from NOVUS; Recombinant Human PKR Protein (LS-G22902-20) from LSBio.

    Techniques: Transfection, Control, Knockdown, Western Blot, Phospho-proteomics

    ( A ) Co-IP analysis of interactions among ALIX, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG (control) or FLAG-ALIX were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( B ) (i) Schematic diagram of ALIX mutants used in this study. FL (full length); Bro1 (Bro1 domain); V domain; PRD (proline-rich domain). Numbers, residue positions. (ii) Schematic illustration of the Ca 2+ /ALG-2-induced open conformation of ALIX. ( C ) Co-IP analysis of interactions among ALIX mutants, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG tagged ALIX mutants and Myc-PKR were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( D ) GST pulldown assay of in vitro translated His-tagged PKR and His-tagged PACT with GST, GST-tagged ALIX, with or without GST-tagged ALG2 in the presence of 10 μM CaCl 2 . Quantification of the GST pulldown (the corresponding protein relative to its input) was performed based on three independent experiments. ( E ) Co-IP analysis of interactions between FLAG-PKR and PACT in HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ) during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( F ) Co-IP analysis of interactions between PKR and GFP-PACT in HEK293T cells transfected with FLAG, or FLAG-ALIX during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-GFP antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 1 mM LLOMe for 30 min. Quantification of LysoIP analysis based on three independent experiments. ( H ) Schematic summary of the findings in Figs. 5 and . See also Fig. . † p ≥ 0.05 (not significant), * p < 0.05, ** p < 0.01, ANOVA. .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) Co-IP analysis of interactions among ALIX, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG (control) or FLAG-ALIX were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( B ) (i) Schematic diagram of ALIX mutants used in this study. FL (full length); Bro1 (Bro1 domain); V domain; PRD (proline-rich domain). Numbers, residue positions. (ii) Schematic illustration of the Ca 2+ /ALG-2-induced open conformation of ALIX. ( C ) Co-IP analysis of interactions among ALIX mutants, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG tagged ALIX mutants and Myc-PKR were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( D ) GST pulldown assay of in vitro translated His-tagged PKR and His-tagged PACT with GST, GST-tagged ALIX, with or without GST-tagged ALG2 in the presence of 10 μM CaCl 2 . Quantification of the GST pulldown (the corresponding protein relative to its input) was performed based on three independent experiments. ( E ) Co-IP analysis of interactions between FLAG-PKR and PACT in HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ) during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( F ) Co-IP analysis of interactions between PKR and GFP-PACT in HEK293T cells transfected with FLAG, or FLAG-ALIX during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-GFP antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 1 mM LLOMe for 30 min. Quantification of LysoIP analysis based on three independent experiments. ( H ) Schematic summary of the findings in Figs. 5 and . See also Fig. . † p ≥ 0.05 (not significant), * p < 0.05, ** p < 0.01, ANOVA. .

    Article Snippet: Recombinant Human ALIX protein (ab132534) from Abcam; Recombinant Human ALG2 Protein (H00085365-P01), Recombinant GST Epitope Tag Protein (NBC1-18537), Recombinant Human PACT His Protein (NBP2-51787) from NOVUS; Recombinant Human PKR Protein (LS-G22902-20) from LSBio.

    Techniques: Co-Immunoprecipitation Assay, Expressing, Control, Immunoprecipitation, Residue, GST Pulldown Assay, In Vitro, Transfection, Knockdown, Purification

    ( A ) AlphaFold 2 predicted the interaction between PKR and ALIX, with the C-terminal PRD domain removed. ( B ) AlphaFold 2 predicted the interaction between PACT and ALIX, with the C-terminal PRD domain removed. ( C ) GST pulldown assay of in vitro translated His-tagged PKR with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( D ) GST pulldown assay of in vitro translated His-tagged PACT with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( E ) Confocal microscopy imaging of GFP-PKR/PACT and ALIX in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( F ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), PKR siRNA for knockdown (PKR KD ), or PACT siRNA for knockdown (PACT KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 1 mM LLOMe for 1 h. NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ANOVA. See also Fig. .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) AlphaFold 2 predicted the interaction between PKR and ALIX, with the C-terminal PRD domain removed. ( B ) AlphaFold 2 predicted the interaction between PACT and ALIX, with the C-terminal PRD domain removed. ( C ) GST pulldown assay of in vitro translated His-tagged PKR with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( D ) GST pulldown assay of in vitro translated His-tagged PACT with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( E ) Confocal microscopy imaging of GFP-PKR/PACT and ALIX in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( F ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), PKR siRNA for knockdown (PKR KD ), or PACT siRNA for knockdown (PACT KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 1 mM LLOMe for 1 h. NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ANOVA. See also Fig. .

    Article Snippet: Recombinant Human ALIX protein (ab132534) from Abcam; Recombinant Human ALG2 Protein (H00085365-P01), Recombinant GST Epitope Tag Protein (NBC1-18537), Recombinant Human PACT His Protein (NBP2-51787) from NOVUS; Recombinant Human PKR Protein (LS-G22902-20) from LSBio.

    Techniques: GST Pulldown Assay, In Vitro, Confocal Microscopy, Imaging, Transfection, Control, Knockdown, Purification

    Reagents and tools table

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: Reagents and tools table

    Article Snippet: Recombinant Human ALIX protein (ab132534) from Abcam; Recombinant Human ALG2 Protein (H00085365-P01), Recombinant GST Epitope Tag Protein (NBC1-18537), Recombinant Human PACT His Protein (NBP2-51787) from NOVUS; Recombinant Human PKR Protein (LS-G22902-20) from LSBio.

    Techniques: Recombinant, Sequencing, Mutagenesis, Control, CRISPR, Staining, Magnetic Beads, Transfection, Lysis, Cytotoxicity Assay, Protease Inhibitor, Software

    ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. ( B ) Immunoblot analysis of phosphorylation of eIF2α (S51) and PKR (T446) in U2OS cells transfected with either scrambled siRNA as control (SCR) or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) and PKR (T446) was quantified based on three independent experiments. ( C ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( D ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells transfected with scrambled siRNA as control (SCR), ALIX siRNA for knockdown (ALIX KD ) or TSG101 siRNA for knockdown (TSG101 KD ). Cells were treated with 2 mM LLOMe for 30 min. The level of phosphorylation of eIF2α (S51) was quantified based on three independent experiments. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells pre-treated with 15 µM BAPTA-AM for 1 h, subjected to 2 mM LLOMe treatment for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i) and was quantified based on three independent experiments. ( G ) Schematic summary of the findings in Figs. 4 and . NT, untreated cells. CTR, control. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. . .

    Article Snippet: Rabbit ALG2 , Proteintech , 15092-1-AP.

    Techniques: Transfection, Control, Knockdown, Western Blot, Phospho-proteomics

    ( A ) Quantification by HCM of LAMP2 in U2OS cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). White masks, algorithm-defined cell boundaries; green masks, computer-identified LAMP2 puncta. ( B ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ), subjected to 2 mM LLOMe treatment for 30 min. ( D ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ), or pre-treated with 15 µM BAPTA-AM for 1 h. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALG2 knockdown cells (ALG2 KD ) overexpressing FLAG or FLAG-ALG2. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) Quantification by HCM of LAMP2 in U2OS cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). White masks, algorithm-defined cell boundaries; green masks, computer-identified LAMP2 puncta. ( B ) Quantification by HCM of G3BP1 puncta in U2OS cells transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; red masks, computer-identified G3BP1 puncta. ( C ) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS transfected with scrambled siRNA as control (SCR), CHMP2B siRNA for knockdown (CHMP2B KD ) or CHMP4B siRNA for knockdown (CHMP4B KD ), subjected to 2 mM LLOMe treatment for 30 min. ( D ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), or ALG2 siRNA for knockdown (ALG2 KD ), or pre-treated with 15 µM BAPTA-AM for 1 h. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( E ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). ( F ) (i) Quantification by HCM of G3BP1 puncta in U2OS ALG2 knockdown cells (ALG2 KD ) overexpressing FLAG or FLAG-ALG2. Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified G3BP1 puncta. (ii) Immunoblot analysis of phosphorylation of eIF2α (S51) in U2OS cells as described in (i). NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ** p < 0.01, ANOVA. See also Fig. .

    Article Snippet: Rabbit ALG2 , Proteintech , 15092-1-AP.

    Techniques: Transfection, Control, Knockdown, Western Blot, Phospho-proteomics

    ( A ) Co-IP analysis of interactions among ALIX, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG (control) or FLAG-ALIX were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( B ) (i) Schematic diagram of ALIX mutants used in this study. FL (full length); Bro1 (Bro1 domain); V domain; PRD (proline-rich domain). Numbers, residue positions. (ii) Schematic illustration of the Ca 2+ /ALG-2-induced open conformation of ALIX. ( C ) Co-IP analysis of interactions among ALIX mutants, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG tagged ALIX mutants and Myc-PKR were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( D ) GST pulldown assay of in vitro translated His-tagged PKR and His-tagged PACT with GST, GST-tagged ALIX, with or without GST-tagged ALG2 in the presence of 10 μM CaCl 2 . Quantification of the GST pulldown (the corresponding protein relative to its input) was performed based on three independent experiments. ( E ) Co-IP analysis of interactions between FLAG-PKR and PACT in HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ) during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( F ) Co-IP analysis of interactions between PKR and GFP-PACT in HEK293T cells transfected with FLAG, or FLAG-ALIX during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-GFP antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 1 mM LLOMe for 30 min. Quantification of LysoIP analysis based on three independent experiments. ( H ) Schematic summary of the findings in Figs. 5 and . See also Fig. . † p ≥ 0.05 (not significant), * p < 0.05, ** p < 0.01, ANOVA. .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) Co-IP analysis of interactions among ALIX, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG (control) or FLAG-ALIX were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( B ) (i) Schematic diagram of ALIX mutants used in this study. FL (full length); Bro1 (Bro1 domain); V domain; PRD (proline-rich domain). Numbers, residue positions. (ii) Schematic illustration of the Ca 2+ /ALG-2-induced open conformation of ALIX. ( C ) Co-IP analysis of interactions among ALIX mutants, PKR and PACT during lysosomal damage. HEK293T cells expressing FLAG tagged ALIX mutants and Myc-PKR were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( D ) GST pulldown assay of in vitro translated His-tagged PKR and His-tagged PACT with GST, GST-tagged ALIX, with or without GST-tagged ALG2 in the presence of 10 μM CaCl 2 . Quantification of the GST pulldown (the corresponding protein relative to its input) was performed based on three independent experiments. ( E ) Co-IP analysis of interactions between FLAG-PKR and PACT in HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ) during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( F ) Co-IP analysis of interactions between PKR and GFP-PACT in HEK293T cells transfected with FLAG, or FLAG-ALIX during lysosomal damage. Cells were treated with 1 mM LLOMe for 30 min. Cell lysates were immunoprecipitated with anti-GFP antibody and immunoblotted for indicated proteins. Quantification of IP analysis based on three independent experiments. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T cells transfected with scrambled siRNA as control (SCR), or ALIX siRNA for knockdown (ALIX KD ). Cells were treated with 1 mM LLOMe for 30 min. Quantification of LysoIP analysis based on three independent experiments. ( H ) Schematic summary of the findings in Figs. 5 and . See also Fig. . † p ≥ 0.05 (not significant), * p < 0.05, ** p < 0.01, ANOVA. .

    Article Snippet: Rabbit ALG2 , Proteintech , 15092-1-AP.

    Techniques: Co-Immunoprecipitation Assay, Expressing, Control, Immunoprecipitation, Residue, GST Pulldown Assay, In Vitro, Transfection, Knockdown, Purification

    ( A ) AlphaFold 2 predicted the interaction between PKR and ALIX, with the C-terminal PRD domain removed. ( B ) AlphaFold 2 predicted the interaction between PACT and ALIX, with the C-terminal PRD domain removed. ( C ) GST pulldown assay of in vitro translated His-tagged PKR with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( D ) GST pulldown assay of in vitro translated His-tagged PACT with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( E ) Confocal microscopy imaging of GFP-PKR/PACT and ALIX in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( F ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), PKR siRNA for knockdown (PKR KD ), or PACT siRNA for knockdown (PACT KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 1 mM LLOMe for 1 h. NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ANOVA. See also Fig. .

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: ( A ) AlphaFold 2 predicted the interaction between PKR and ALIX, with the C-terminal PRD domain removed. ( B ) AlphaFold 2 predicted the interaction between PACT and ALIX, with the C-terminal PRD domain removed. ( C ) GST pulldown assay of in vitro translated His-tagged PKR with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( D ) GST pulldown assay of in vitro translated His-tagged PACT with GST or GST-tagged ALIX (i) or ALG2 (ii) in the presence of 10 μM CaCl 2 . ( E ) Confocal microscopy imaging of GFP-PKR/PACT and ALIX in U2OS cells treated with 2 mM LLOMe for 30 min. Scale bar, 5 μm. ( F ) Quantification by HCM of ALIX puncta in U2OS cells transfected with scrambled siRNA as control (SCR), PKR siRNA for knockdown (PKR KD ), or PACT siRNA for knockdown (PACT KD ). Cells were treated with 2 mM LLOMe for 30 min. White masks, algorithm-defined cell boundaries; green masks, computer-identified ALIX puncta. ( G ) Analysis of proteins associated with purified lysosomes (LysoIP; TMEM192-3xHA) from HEK293T ALIX knockdown cells (ALIX KD ) overexpressing FLAG or FLAG-ALIX. Cells were treated with 1 mM LLOMe for 1 h. NT, untreated cells. Data, means ± SEM ( n = 3); HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). † p ≥ 0.05 (not significant), ANOVA. See also Fig. .

    Article Snippet: Rabbit ALG2 , Proteintech , 15092-1-AP.

    Techniques: GST Pulldown Assay, In Vitro, Confocal Microscopy, Imaging, Transfection, Control, Knockdown, Purification

    Reagents and tools table

    Journal: The EMBO Journal

    Article Title: Calcium signaling from damaged lysosomes induces cytoprotective stress granules

    doi: 10.1038/s44318-024-00292-1

    Figure Lengend Snippet: Reagents and tools table

    Article Snippet: Rabbit ALG2 , Proteintech , 15092-1-AP.

    Techniques: Recombinant, Sequencing, Mutagenesis, Control, CRISPR, Staining, Magnetic Beads, Transfection, Lysis, Cytotoxicity Assay, Protease Inhibitor, Software