mitochondria isolation buffer Search Results


mitochondria isolation buffer  (Bellco Glass)
90
Bellco Glass mitochondria isolation buffer
Mitochondria Isolation Buffer, supplied by Bellco Glass, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mitochondria isolation buffer - by Bioz Stars, 2026-04
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mitochondria isolation buffer (mib: sucrose 275mm)  (Euromedex)
90
Euromedex mitochondria isolation buffer (mib: sucrose 275mm)
Mitochondria Isolation Buffer (Mib: Sucrose 275mm), supplied by Euromedex, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mitochondria isolation buffer (mib: sucrose 275mm) - by Bioz Stars, 2026-04
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lysis buffer qproteome mitochondria isolation kit  (Qiagen)
90
Qiagen lysis buffer qproteome mitochondria isolation kit
Lysis Buffer Qproteome Mitochondria Isolation Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lysis buffer qproteome mitochondria isolation kit - by Bioz Stars, 2026-04
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mitochondria isolation buffer (mib: 10 mm tris/mops, ph 7.4, 1 mm egta/tris, 200 mm sucrose)  (Thomas Scientific)
90
Thomas Scientific mitochondria isolation buffer (mib: 10 mm tris/mops, ph 7.4, 1 mm egta/tris, 200 mm sucrose)
Mitochondria Isolation Buffer (Mib: 10 Mm Tris/Mops, Ph 7.4, 1 Mm Egta/Tris, 200 Mm Sucrose), supplied by Thomas Scientific, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mitochondria isolation buffer (mib: 10 mm tris/mops, ph 7.4, 1 mm egta/tris, 200 mm sucrose)/product/Thomas Scientific
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mitochondria isolation buffer (mib: 10 mm tris/mops, ph 7.4, 1 mm egta/tris, 200 mm sucrose) - by Bioz Stars, 2026-04
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mitochondria isolation buffer  (Kontes Glass)
90
Kontes Glass mitochondria isolation buffer
Mitochondria Isolation Buffer, supplied by Kontes Glass, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mitochondria isolation buffer/product/Kontes Glass
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mitochondria isolation buffer - by Bioz Stars, 2026-04
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cell lysis buffer for western, ip, and cell mitochondria isolation kit  (Beyotime)
90
Beyotime cell lysis buffer for western, ip, and cell mitochondria isolation kit
Cell Lysis Buffer For Western, Ip, And Cell Mitochondria Isolation Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cell lysis buffer for western, ip, and cell mitochondria isolation kit/product/Beyotime
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cell lysis buffer for western, ip, and cell mitochondria isolation kit - by Bioz Stars, 2026-04
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cell mitochondria isolation buffer  (Beyotime)
90
Beyotime cell mitochondria isolation buffer
MUL1 regulated the translocation of HSPA9 from <t>mitochondria</t> to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.
Cell Mitochondria Isolation Buffer, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cell mitochondria isolation buffer/product/Beyotime
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cell mitochondria isolation buffer - by Bioz Stars, 2026-04
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mitochondria isolation buffer  (Hausser Scientific)
90
Hausser Scientific mitochondria isolation buffer
MUL1 regulated the translocation of HSPA9 from <t>mitochondria</t> to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.
Mitochondria Isolation Buffer, supplied by Hausser Scientific, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mitochondria isolation buffer/product/Hausser Scientific
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mitochondria isolation buffer - by Bioz Stars, 2026-04
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1 ml mitochondria isolation buffer  (BioExpress)
90
BioExpress 1 ml mitochondria isolation buffer
MUL1 regulated the translocation of HSPA9 from <t>mitochondria</t> to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.
1 Ml Mitochondria Isolation Buffer, supplied by BioExpress, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1 ml mitochondria isolation buffer/product/BioExpress
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1 ml mitochondria isolation buffer - by Bioz Stars, 2026-04
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cell mitochondria isolation buffer containing pmsf  (Beyotime)
90
Beyotime cell mitochondria isolation buffer containing pmsf
MUL1 regulated the translocation of HSPA9 from <t>mitochondria</t> to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.
Cell Mitochondria Isolation Buffer Containing Pmsf, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cell mitochondria isolation buffer containing pmsf/product/Beyotime
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cell mitochondria isolation buffer containing pmsf - by Bioz Stars, 2026-04
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mitochondria storage buffer qproteome mitochondria isolation kit  (Qiagen)
90
Qiagen mitochondria storage buffer qproteome mitochondria isolation kit
MUL1 regulated the translocation of HSPA9 from <t>mitochondria</t> to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.
Mitochondria Storage Buffer Qproteome Mitochondria Isolation Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mitochondria storage buffer qproteome mitochondria isolation kit/product/Qiagen
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mitochondria storage buffer qproteome mitochondria isolation kit - by Bioz Stars, 2026-04
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9 ml ice-cold mitochondria isolation buffer (225 mmol/l mannitol, 75 mmol/l sucrose, and 2 mmol/l k 2 hpo 4 [ph 7.2])  (Kontes Glass)
90
Kontes Glass 9 ml ice-cold mitochondria isolation buffer (225 mmol/l mannitol, 75 mmol/l sucrose, and 2 mmol/l k 2 hpo 4 [ph 7.2])
MUL1 regulated the translocation of HSPA9 from <t>mitochondria</t> to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.
9 Ml Ice Cold Mitochondria Isolation Buffer (225 Mmol/L Mannitol, 75 Mmol/L Sucrose, And 2 Mmol/L K 2 Hpo 4 [Ph 7.2]), supplied by Kontes Glass, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/9 ml ice-cold mitochondria isolation buffer (225 mmol/l mannitol, 75 mmol/l sucrose, and 2 mmol/l k 2 hpo 4 [ph 7.2])/product/Kontes Glass
Average 90 stars, based on 1 article reviews
9 ml ice-cold mitochondria isolation buffer (225 mmol/l mannitol, 75 mmol/l sucrose, and 2 mmol/l k 2 hpo 4 [ph 7.2]) - by Bioz Stars, 2026-04
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Image Search Results


MUL1 regulated the translocation of HSPA9 from mitochondria to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.

Journal: International Journal of Biological Sciences

Article Title: SUMO E3 ligase MUL1 inhibits lymph node metastasis of bladder cancer by mediating mitochondrial HSPA9 translocation

doi: 10.7150/ijbs.98772

Figure Lengend Snippet: MUL1 regulated the translocation of HSPA9 from mitochondria to nucleus. (A). Identification of MUL1-interacting proteins via MS analysis is shown in Venn diagrams. HSPA9 was one of the 103 overlapping proteins present in both the T24 and the UM-UC-3 MS analysis results. (B). Anti-MUL1 immunoprecipitation (IP) was performed, and HSPA9 was detected via western blotting. Input served as a positive control, and IgG served as a negative control. (C). Representative immunofluorescence (IF) images of MUL1 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: MUL1. Scale bars are shown in the right corner of the images. (D). Representative IF images of TOM20 and HSPA9 colocalization in T24 and UM-UC-3 cells. Green: HSPA9; red: TOM20. Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E-F). Mitochondrial, cytosolic and whole-cell lysates of cells with stable MUL1 knockdown or overexpression were prepared. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (G). Nuclear and mitochondria-free cytoplasmic faction of MUL1-knockdown and -overexpression cells were separated. SUMO-HSPA9 and HSPA9 levels were detected via western blotting. Lamin B1 was used as a reference for nuclear proteins and α-tubulin was used as a reference for mitochondria-free cytoplasmic proteins.

Article Snippet: Cells were suspended in 2 ml of cell mitochondria isolation buffer containing 100 mM PMSF (ST506, Beyotime) for 30 minutes.

Techniques: Translocation Assay, Immunoprecipitation, Western Blot, Positive Control, Negative Control, Immunofluorescence, Knockdown, Over Expression

MUL1 SUMOylated HSPA9 at the lysine 612 (K612) residue. (A-B). Anti-HSPA9 IP was performed in MUL1-silencing (E) and MUL1-overexpressing (F) cell lysates. SUMO2/3 and HSPA9 levels were detected via western blotting. (C). T24 cells were treated with ML792 at the concentrations indicated in the images. Mitochondrial and cytosolic cell lysates were separated and prepared. HSPA9 levels in mitochondria and mitochondria-free cell fractions were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (D). Representative IF images of HSPA9 localization in T24 cells with the treatment of ML792 are shown. Green: HSPA9; red: TOM20; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E). GPS-SUMO, JASSA and SUMOplot tools were used to predict the HSPA9 SUMOylation site. Sequences around SUMO-conjugation motifs are shown. Red: SUMO-conjugation motifs. (F). Schematic diagram of the predicted HSPA9 K612 site using SWISS-MODEL tools. (G). Wild-type HSPA9 and HSPA9-K612R were overexpressed in T24 and UM-UC-3 cells to generate OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. GFP levels were detected via western blotting. (H). Anti-GFP IP was performed in OE-HSPA9 and OE-HSPA9-K612R cell lysates. SUMO2/3 levels were detected via western blotting. (I). Mitochondrial and mitochondria-free cell fractions of OE-HSPA9 and OE-HSPA9-K612R cells were prepared. GFP expression was detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (J). Stable overexpression of exogenous MUL1 was performed in OE-HSPA9 or OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Representative IF images of GFP and TOM20 colocalization are shown. Green: GFP; red: TOM20; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. -, no statistical significance; *, P < 0.05; **, P < 0.01.

Journal: International Journal of Biological Sciences

Article Title: SUMO E3 ligase MUL1 inhibits lymph node metastasis of bladder cancer by mediating mitochondrial HSPA9 translocation

doi: 10.7150/ijbs.98772

Figure Lengend Snippet: MUL1 SUMOylated HSPA9 at the lysine 612 (K612) residue. (A-B). Anti-HSPA9 IP was performed in MUL1-silencing (E) and MUL1-overexpressing (F) cell lysates. SUMO2/3 and HSPA9 levels were detected via western blotting. (C). T24 cells were treated with ML792 at the concentrations indicated in the images. Mitochondrial and cytosolic cell lysates were separated and prepared. HSPA9 levels in mitochondria and mitochondria-free cell fractions were detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (D). Representative IF images of HSPA9 localization in T24 cells with the treatment of ML792 are shown. Green: HSPA9; red: TOM20; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. (E). GPS-SUMO, JASSA and SUMOplot tools were used to predict the HSPA9 SUMOylation site. Sequences around SUMO-conjugation motifs are shown. Red: SUMO-conjugation motifs. (F). Schematic diagram of the predicted HSPA9 K612 site using SWISS-MODEL tools. (G). Wild-type HSPA9 and HSPA9-K612R were overexpressed in T24 and UM-UC-3 cells to generate OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. GFP levels were detected via western blotting. (H). Anti-GFP IP was performed in OE-HSPA9 and OE-HSPA9-K612R cell lysates. SUMO2/3 levels were detected via western blotting. (I). Mitochondrial and mitochondria-free cell fractions of OE-HSPA9 and OE-HSPA9-K612R cells were prepared. GFP expression was detected via western blotting. TOM70 was used as a reference for mitochondrial proteins, and α-tubulin was used as a reference for cytosolic proteins. (J). Stable overexpression of exogenous MUL1 was performed in OE-HSPA9 or OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Representative IF images of GFP and TOM20 colocalization are shown. Green: GFP; red: TOM20; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. TOM20 was used as a mitochondrial tracer. -, no statistical significance; *, P < 0.05; **, P < 0.01.

Article Snippet: Cells were suspended in 2 ml of cell mitochondria isolation buffer containing 100 mM PMSF (ST506, Beyotime) for 30 minutes.

Techniques: Residue, Western Blot, Conjugation Assay, Expressing, Over Expression

SUMOylated HSPA9 catalyzed the degradation of SUZ12 and EZH2. (A). The identification of HSPA9-interacting proteins is shown in a schematic diagram. By comparing the qualitative MS analysis and relative quantitative proteomics results, SUZ12 and EZH2 were selected for further research. (B). Nuclear and mitochondria-free cytoplasmic faction of T24 and UM-UC-3 cells were separated. Anti-HSPA9 IP was performed in nuclear and mitochondria-free cytoplasmic protein lysates. HSPA9, SUZ12 and EZH2 levels were detected via western blotting. IgG served as a negative control. (C). Anti-GFP IP was performed in OE-HSPA9 and OE-HSPA9-K612R cell lysates. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. SUZ12 and EZH2 levels were detected via western blotting. Input served as a positive control, and IgG served as a negative control. (D). Representative IF images of GFP-SUZ12 and GFP-EZH2 colocalization in T24 OE-HSPA9 and OE-HSPA9-K612R cells are shown. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Green: GFP; red: SUZ12/EZH2; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. (E). Stable overexpression of vector or exogenous MUL1 was performed in T24 OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Representative IF images of GFP and SUZ12 colocalization are shown. Green: GFP; red: SUZ12; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. (F). SUZ12, EZH2, STAT3 and p-STAT3 levels were detected via western blotting in OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. (G). SUZ12, EZH2, STAT3 and p-STAT3 levels were detected via western blotting in MUL1-silencing or MUL1-overexpressing cells. (H). Anti-EZH2 and anti-SUZ12 IP were performed in T24 OE-HSPA9 and OE-HSPA9-K612R cell lysates. Ubiquitin-K48 levels were detected via western blotting.

Journal: International Journal of Biological Sciences

Article Title: SUMO E3 ligase MUL1 inhibits lymph node metastasis of bladder cancer by mediating mitochondrial HSPA9 translocation

doi: 10.7150/ijbs.98772

Figure Lengend Snippet: SUMOylated HSPA9 catalyzed the degradation of SUZ12 and EZH2. (A). The identification of HSPA9-interacting proteins is shown in a schematic diagram. By comparing the qualitative MS analysis and relative quantitative proteomics results, SUZ12 and EZH2 were selected for further research. (B). Nuclear and mitochondria-free cytoplasmic faction of T24 and UM-UC-3 cells were separated. Anti-HSPA9 IP was performed in nuclear and mitochondria-free cytoplasmic protein lysates. HSPA9, SUZ12 and EZH2 levels were detected via western blotting. IgG served as a negative control. (C). Anti-GFP IP was performed in OE-HSPA9 and OE-HSPA9-K612R cell lysates. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. SUZ12 and EZH2 levels were detected via western blotting. Input served as a positive control, and IgG served as a negative control. (D). Representative IF images of GFP-SUZ12 and GFP-EZH2 colocalization in T24 OE-HSPA9 and OE-HSPA9-K612R cells are shown. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Green: GFP; red: SUZ12/EZH2; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. (E). Stable overexpression of vector or exogenous MUL1 was performed in T24 OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. Representative IF images of GFP and SUZ12 colocalization are shown. Green: GFP; red: SUZ12; blue: nuclei (DAPI). Scale bars are shown in the right corner of the images. (F). SUZ12, EZH2, STAT3 and p-STAT3 levels were detected via western blotting in OE-HSPA9 and OE-HSPA9-K612R cells. Both wild-type HSPA9 and HSPA9-K612R were tagged with GFP. (G). SUZ12, EZH2, STAT3 and p-STAT3 levels were detected via western blotting in MUL1-silencing or MUL1-overexpressing cells. (H). Anti-EZH2 and anti-SUZ12 IP were performed in T24 OE-HSPA9 and OE-HSPA9-K612R cell lysates. Ubiquitin-K48 levels were detected via western blotting.

Article Snippet: Cells were suspended in 2 ml of cell mitochondria isolation buffer containing 100 mM PMSF (ST506, Beyotime) for 30 minutes.

Techniques: Western Blot, Negative Control, Positive Control, Over Expression, Plasmid Preparation