alg2  (Santa Cruz Biotechnology)

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

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

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

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

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

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

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

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

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

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