tfeb gfp Search Results


tfeb–gfp  (Lonza)
90
Lonza tfeb–gfp
mTORC1 regulates <t>TFEB.</t> ( A ) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p-T202/Y204-ERK1/2, ERK1/2, p-T389-S6K, and S6K to measure ERK and mTORC1 activities. ( B ) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB–3 × FLAG HeLa cells starved in amino acid-free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies. ( C ) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. <t>TFEB–GFP</t> HeLa cells were seeded in 96-well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin-Elmer). Scale bars represent 30 μm. ( D ) Dose–response curves of the effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 384-well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details). ( E ) Immunofluorescence of HEK-293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM. ( F ) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB–3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh-) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency. ( G ) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB–3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3. ( H ) Binding of TFEB to mTORC1. HEK-293T cells that express TFEB–3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG–Rap2A served as negative control.
Tfeb–Gfp, supplied by Lonza, 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/tfeb–gfp/product/Lonza
Average 90 stars, based on 1 article reviews
tfeb–gfp - by Bioz Stars, 2026-03
90/100 stars
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tfeb-gfp expression vector  (Orbigen Inc)
90
Orbigen Inc tfeb-gfp expression vector
<t>TFEB</t> translocates to the nucleus during mitophagy in a Parkin- and PINK1-dependent manner. (A) YFP-Parkin HeLa cells were treated with O/A for up to 10 h, fractionated, and immunoblotted. (B) Quantification of data in A. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (C) mCherry-Parkin HeLa cells were left untreated (Control), starved (2 h), or treated with torin 1 (2 h), O/A (6 h), or valinomycin (Val; 6 h). CIP treatment of cell lysates was performed before immunoblotting. (D) WT and mCherry-Parkin HeLa cells were treated with DMSO or O/A (6 h), lysed, and immunoblotted. A CIP-treated control was included as a reference for total TFEB dephosphorylation. (E) WT and mCherry-Parkin HeLa cells were treated with DMSO, torin 1, or O/A for 18 h and analyzed by quantitative PCR for TFEB target gene expression. Data are means ± SD ( n = 3). (F) WT and PINK1 KO HeLa cells stably expressing <t>TFEB-GFP</t> with or without mCherry-Parkin were treated as in C. Fixed cells were stained with DAPI and analyzed by immunofluorescence. Bars, 10 µm. See Fig. S1 F for quantification. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Tfeb Gfp Expression Vector, supplied by Orbigen Inc, 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/tfeb-gfp expression vector/product/Orbigen Inc
Average 90 stars, based on 1 article reviews
tfeb-gfp expression vector - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier
s142a serine-to-alanine site mutation of tfeb-gfp  (GenScript corporation)
90
GenScript corporation s142a serine-to-alanine site mutation of tfeb-gfp
<t>TFEB</t> translocates to the nucleus during mitophagy in a Parkin- and PINK1-dependent manner. (A) YFP-Parkin HeLa cells were treated with O/A for up to 10 h, fractionated, and immunoblotted. (B) Quantification of data in A. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (C) mCherry-Parkin HeLa cells were left untreated (Control), starved (2 h), or treated with torin 1 (2 h), O/A (6 h), or valinomycin (Val; 6 h). CIP treatment of cell lysates was performed before immunoblotting. (D) WT and mCherry-Parkin HeLa cells were treated with DMSO or O/A (6 h), lysed, and immunoblotted. A CIP-treated control was included as a reference for total TFEB dephosphorylation. (E) WT and mCherry-Parkin HeLa cells were treated with DMSO, torin 1, or O/A for 18 h and analyzed by quantitative PCR for TFEB target gene expression. Data are means ± SD ( n = 3). (F) WT and PINK1 KO HeLa cells stably expressing <t>TFEB-GFP</t> with or without mCherry-Parkin were treated as in C. Fixed cells were stained with DAPI and analyzed by immunofluorescence. Bars, 10 µm. See Fig. S1 F for quantification. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
S142a Serine To Alanine Site Mutation Of Tfeb Gfp, supplied by GenScript corporation, 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/s142a serine-to-alanine site mutation of tfeb-gfp/product/GenScript corporation
Average 90 stars, based on 1 article reviews
s142a serine-to-alanine site mutation of tfeb-gfp - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier

Image Search Results


mTORC1 regulates TFEB. ( A ) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p-T202/Y204-ERK1/2, ERK1/2, p-T389-S6K, and S6K to measure ERK and mTORC1 activities. ( B ) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB–3 × FLAG HeLa cells starved in amino acid-free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies. ( C ) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 96-well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin-Elmer). Scale bars represent 30 μm. ( D ) Dose–response curves of the effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 384-well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details). ( E ) Immunofluorescence of HEK-293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM. ( F ) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB–3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh-) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency. ( G ) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB–3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3. ( H ) Binding of TFEB to mTORC1. HEK-293T cells that express TFEB–3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG–Rap2A served as negative control.

Journal: The EMBO Journal

Article Title: A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

doi: 10.1038/emboj.2012.32

Figure Lengend Snippet: mTORC1 regulates TFEB. ( A ) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p-T202/Y204-ERK1/2, ERK1/2, p-T389-S6K, and S6K to measure ERK and mTORC1 activities. ( B ) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB–3 × FLAG HeLa cells starved in amino acid-free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies. ( C ) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 96-well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin-Elmer). Scale bars represent 30 μm. ( D ) Dose–response curves of the effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 384-well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details). ( E ) Immunofluorescence of HEK-293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM. ( F ) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB–3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh-) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency. ( G ) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB–3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3. ( H ) Binding of TFEB to mTORC1. HEK-293T cells that express TFEB–3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG–Rap2A served as negative control.

Article Snippet: MEFs were transiently transfected with TFEB–GFP and mRFP–Rab7 by nucleofection (Lonza).

Techniques: Western Blot, Isolation, De-Phosphorylation Assay, Translocation Assay, Fractionation, Cell Culture, Microscopy, Concentration Assay, Software, Immunofluorescence, Staining, Stable Transfection, Expressing, Infection, Luciferase, Binding Assay, Immunoprecipitation, Negative Control

mTORC1 binds and phosphorylates TFEB on the lysosomal surface. ( A ) Spinning disk confocal image of a MEF cell that co-expresses TFEB–GFP and mRFP–Rab7 (green and red in the merge, respectively). ( B ) Time-lapse of TFEB- and Rab7-positive lysosomes from the boxed region in ( A ). Time intervals are in seconds. ( C ) Time-lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB–GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1-induced lysosomal accumulation of TFEB–GFP. Time intervals are in minutes. ( D ) Immunofluorescence of HEK-293T cells expressing TFEB–3 × FLAG, treated with DMSO ( top ) or Torin1 ( bottom ) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). ( E ) FRAP analysis of TFEB–GFP-positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots. ( F ) Time-lapse of photobleaching and fluorescence recovery of TFEB–GFP-positive lysosomes from control-treated MEFs ( top ) or MEFs treated with Torin 1 ( bottom ). Red arrowheads indicate time of photobleaching. Time intervals are in seconds. ( G ) Torin 1 increases binding of TFEB to mTORC1. HEK-293T cells that express TFEB–3 × FLAG along with HAGST-Rap2A or HAGST-Rags DN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG–Metap2 served as negative control. ( H ) Immunofluorescence of HEK-293T cells that express TFEB–3 × FLAG along with Rap2A ( top ) or the Rags DN mutants ( bottom ), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.

Journal: The EMBO Journal

Article Title: A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

doi: 10.1038/emboj.2012.32

Figure Lengend Snippet: mTORC1 binds and phosphorylates TFEB on the lysosomal surface. ( A ) Spinning disk confocal image of a MEF cell that co-expresses TFEB–GFP and mRFP–Rab7 (green and red in the merge, respectively). ( B ) Time-lapse of TFEB- and Rab7-positive lysosomes from the boxed region in ( A ). Time intervals are in seconds. ( C ) Time-lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB–GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1-induced lysosomal accumulation of TFEB–GFP. Time intervals are in minutes. ( D ) Immunofluorescence of HEK-293T cells expressing TFEB–3 × FLAG, treated with DMSO ( top ) or Torin1 ( bottom ) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). ( E ) FRAP analysis of TFEB–GFP-positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots. ( F ) Time-lapse of photobleaching and fluorescence recovery of TFEB–GFP-positive lysosomes from control-treated MEFs ( top ) or MEFs treated with Torin 1 ( bottom ). Red arrowheads indicate time of photobleaching. Time intervals are in seconds. ( G ) Torin 1 increases binding of TFEB to mTORC1. HEK-293T cells that express TFEB–3 × FLAG along with HAGST-Rap2A or HAGST-Rags DN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG–Metap2 served as negative control. ( H ) Immunofluorescence of HEK-293T cells that express TFEB–3 × FLAG along with Rap2A ( top ) or the Rags DN mutants ( bottom ), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.

Article Snippet: MEFs were transiently transfected with TFEB–GFP and mRFP–Rab7 by nucleofection (Lonza).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Binding Assay, Immunoprecipitation, Western Blot, Negative Control

TFEB translocates to the nucleus during mitophagy in a Parkin- and PINK1-dependent manner. (A) YFP-Parkin HeLa cells were treated with O/A for up to 10 h, fractionated, and immunoblotted. (B) Quantification of data in A. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (C) mCherry-Parkin HeLa cells were left untreated (Control), starved (2 h), or treated with torin 1 (2 h), O/A (6 h), or valinomycin (Val; 6 h). CIP treatment of cell lysates was performed before immunoblotting. (D) WT and mCherry-Parkin HeLa cells were treated with DMSO or O/A (6 h), lysed, and immunoblotted. A CIP-treated control was included as a reference for total TFEB dephosphorylation. (E) WT and mCherry-Parkin HeLa cells were treated with DMSO, torin 1, or O/A for 18 h and analyzed by quantitative PCR for TFEB target gene expression. Data are means ± SD ( n = 3). (F) WT and PINK1 KO HeLa cells stably expressing TFEB-GFP with or without mCherry-Parkin were treated as in C. Fixed cells were stained with DAPI and analyzed by immunofluorescence. Bars, 10 µm. See Fig. S1 F for quantification. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: TFEB translocates to the nucleus during mitophagy in a Parkin- and PINK1-dependent manner. (A) YFP-Parkin HeLa cells were treated with O/A for up to 10 h, fractionated, and immunoblotted. (B) Quantification of data in A. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (C) mCherry-Parkin HeLa cells were left untreated (Control), starved (2 h), or treated with torin 1 (2 h), O/A (6 h), or valinomycin (Val; 6 h). CIP treatment of cell lysates was performed before immunoblotting. (D) WT and mCherry-Parkin HeLa cells were treated with DMSO or O/A (6 h), lysed, and immunoblotted. A CIP-treated control was included as a reference for total TFEB dephosphorylation. (E) WT and mCherry-Parkin HeLa cells were treated with DMSO, torin 1, or O/A for 18 h and analyzed by quantitative PCR for TFEB target gene expression. Data are means ± SD ( n = 3). (F) WT and PINK1 KO HeLa cells stably expressing TFEB-GFP with or without mCherry-Parkin were treated as in C. Fixed cells were stained with DAPI and analyzed by immunofluorescence. Bars, 10 µm. See Fig. S1 F for quantification. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Expressing, Western Blot, De-Phosphorylation Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Staining, Immunofluorescence

Analysis of Parkin-dependent effects on mTORC1 activity and TFEB association with 14-3-3 proteins. (A and C) WT and YFP-Parkin HeLa cells were treated with DMSO, O/A, or torin 1 as indicated, lysed, and immunoblotted. (B and D) Quantification of data in A and C, respectively. Protein levels were normalized to actin and the ratio of phosphorylated to total protein in treated samples is expressed relative to DMSO controls. Data are means ± SD ( n = 3); no differences observed were statistically significant. (E) HeLa cells stably expressing TFEB-GFP were transfected with control or untagged Parkin DNA and treated the next day with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Cells were lysed and TFEB-GFP was immunoprecipitated with anti-GFP beads. Cell lysates (Input) and immunoprecipitated proteins were immunoblotted. Images are representative of n = 2 experiments.

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Analysis of Parkin-dependent effects on mTORC1 activity and TFEB association with 14-3-3 proteins. (A and C) WT and YFP-Parkin HeLa cells were treated with DMSO, O/A, or torin 1 as indicated, lysed, and immunoblotted. (B and D) Quantification of data in A and C, respectively. Protein levels were normalized to actin and the ratio of phosphorylated to total protein in treated samples is expressed relative to DMSO controls. Data are means ± SD ( n = 3); no differences observed were statistically significant. (E) HeLa cells stably expressing TFEB-GFP were transfected with control or untagged Parkin DNA and treated the next day with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Cells were lysed and TFEB-GFP was immunoprecipitated with anti-GFP beads. Cell lysates (Input) and immunoprecipitated proteins were immunoblotted. Images are representative of n = 2 experiments.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation

Parkin acts upstream of Rag GTPases to regulate TFEB subcellular localization. (A and B) HeLa cells stably expressing TFEB-GFP with (B) or without (A) mCherry-Parkin were transfected with empty vector or active or inactive RagB/D heterodimer DNA (detected with αHA antibody). The indicated cells were starved (2 h) or treated with DMSO or O/A (6 h) before fixation and immunofluorescence analysis. Images are representative of n = 2 experiments. 75–100% of cells observed (mean of 175 cells per condition) exhibited the given phenotype. Asterisks indicate αHA-negative cells. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Parkin acts upstream of Rag GTPases to regulate TFEB subcellular localization. (A and B) HeLa cells stably expressing TFEB-GFP with (B) or without (A) mCherry-Parkin were transfected with empty vector or active or inactive RagB/D heterodimer DNA (detected with αHA antibody). The indicated cells were starved (2 h) or treated with DMSO or O/A (6 h) before fixation and immunofluorescence analysis. Images are representative of n = 2 experiments. 75–100% of cells observed (mean of 175 cells per condition) exhibited the given phenotype. Asterisks indicate αHA-negative cells. Bars, 10 µm.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunofluorescence

Atg5 and Atg9A are required for Parkin-mediated TFEB translocation. (A) WT and Atg5 KO cells stably expressing mCherry-Parkin were treated with DMSO or O/A (6 h), fixed, immunostained for TFEB, and analyzed by immunofluorescence. Bars, 10 µm. (B) Quantification of endogenous TFEB nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) Untreated WT and Atg5 KO cells stably expressing mCherry-Parkin and GFP-Atg5 as indicated were lysed and immunoblotted. (D) Cells from C were treated with DMSO or O/A (6 h), lysed, fractionated, and immunoblotted. (E) Quantification of data in D. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB was expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (F) WT and Atg5 KO HeLa cells stably expressing mCherry-Parkin as indicated were treated with DMSO (6 h), torin 1 (2 h), CIP (1 h), and O/A (6 h) as indicated, lysed, and immunoblotted. Images are representative of n = 3 experiments. (G) WT and Atg5 KO cells expressing mCherry-Parkin as indicated were starved (2 h) or left untreated, lysed, fractionated, and immunoblotted. Images are representative of n = 3 experiments. (H) WT and Atg9A KO HeLa cells expressing mCherry-Parkin as indicated were starved (2 h), or treated with DMSO (Ctrl) or O/A for 6 h. Cell lysates were processed as in D. (I) Quantification of endogenous TFEB nuclear localization in H, performed as in E. Data are means ± SD ( n = 3). C, cytosol; N, nuclear. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Atg5 and Atg9A are required for Parkin-mediated TFEB translocation. (A) WT and Atg5 KO cells stably expressing mCherry-Parkin were treated with DMSO or O/A (6 h), fixed, immunostained for TFEB, and analyzed by immunofluorescence. Bars, 10 µm. (B) Quantification of endogenous TFEB nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) Untreated WT and Atg5 KO cells stably expressing mCherry-Parkin and GFP-Atg5 as indicated were lysed and immunoblotted. (D) Cells from C were treated with DMSO or O/A (6 h), lysed, fractionated, and immunoblotted. (E) Quantification of data in D. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB was expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (F) WT and Atg5 KO HeLa cells stably expressing mCherry-Parkin as indicated were treated with DMSO (6 h), torin 1 (2 h), CIP (1 h), and O/A (6 h) as indicated, lysed, and immunoblotted. Images are representative of n = 3 experiments. (G) WT and Atg5 KO cells expressing mCherry-Parkin as indicated were starved (2 h) or left untreated, lysed, fractionated, and immunoblotted. Images are representative of n = 3 experiments. (H) WT and Atg9A KO HeLa cells expressing mCherry-Parkin as indicated were starved (2 h), or treated with DMSO (Ctrl) or O/A for 6 h. Cell lysates were processed as in D. (I) Quantification of endogenous TFEB nuclear localization in H, performed as in E. Data are means ± SD ( n = 3). C, cytosol; N, nuclear. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Translocation Assay, Stable Transfection, Expressing, Immunofluorescence, Fluorescence

Parkin- and Atg5-dependent regulation of TFEB homologue subcellular localization. (A) WT HeLa cells stably expressing mCherry-Parkin, TFE3-GFP, MITF1-GFP, and TFEC-YFP as indicated were treated with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Fixed cells were analyzed by immunofluorescence. (B) Quantification of ectopic TFE3, MITF1, and TFEC nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) WT and Atg5 KO cells stably expressing mCherry-Parkin treated with DMSO or O/A (6 h) were fixed, immunostained for TFE3 or MITF, and analyzed by immunofluorescence. (D) Quantification of endogenous TFE3 and MITF nuclear localization in C. Analysis was performed as in B (40–60 cells/field, 4 fields/condition, n = 3 experiments). Data are means ± SD. For all graphs: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Parkin- and Atg5-dependent regulation of TFEB homologue subcellular localization. (A) WT HeLa cells stably expressing mCherry-Parkin, TFE3-GFP, MITF1-GFP, and TFEC-YFP as indicated were treated with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Fixed cells were analyzed by immunofluorescence. (B) Quantification of ectopic TFE3, MITF1, and TFEC nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) WT and Atg5 KO cells stably expressing mCherry-Parkin treated with DMSO or O/A (6 h) were fixed, immunostained for TFE3 or MITF, and analyzed by immunofluorescence. (D) Quantification of endogenous TFE3 and MITF nuclear localization in C. Analysis was performed as in B (40–60 cells/field, 4 fields/condition, n = 3 experiments). Data are means ± SD. For all graphs: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Fluorescence