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Addgene inc ha-tagged sqstm1

Palmostatin B alters autophagy in HeLa cells, assessed through LC3B and <t>SQSTM1</t> immunoblotting. HeLa cells were treated with 0.1% DMSO or 100 μM palmostatin B (PalmB) for 18 h. Autophagy was induced by 4 h EBSS treatment or induced and inhibited by 4 h EBSS/2 h treatment with 500 nM BafA1. (A) Representative images of immunoblotting conducted for autophagy markers SQSTM1 and LC3‐II with and without Palm B treatment and autophagy induction. (B) Quantification of immunoblots for SQSTM1 normalized to ACTB/β‐actin. Densitometry was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA. (C) Quantification of immunoblots for LC3‐II normalized to ACTB. Quantification was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA).

Ha Tagged Sqstm1, supplied by Addgene inc, 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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ha-tagged sqstm1 - by Bioz Stars, 2026-03

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1) Product Images from "Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy"

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

Journal: The FASEB Journal

doi: 10.1096/fj.202401781R

Palmostatin B alters autophagy in HeLa cells, assessed through LC3B and SQSTM1 immunoblotting. HeLa cells were treated with 0.1% DMSO or 100 μM palmostatin B (PalmB) for 18 h. Autophagy was induced by 4 h EBSS treatment or induced and inhibited by 4 h EBSS/2 h treatment with 500 nM BafA1. (A) Representative images of immunoblotting conducted for autophagy markers SQSTM1 and LC3‐II with and without Palm B treatment and autophagy induction. (B) Quantification of immunoblots for SQSTM1 normalized to ACTB/β‐actin. Densitometry was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA. (C) Quantification of immunoblots for LC3‐II normalized to ACTB. Quantification was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA).

Figure Legend Snippet: Palmostatin B alters autophagy in HeLa cells, assessed through LC3B and SQSTM1 immunoblotting. HeLa cells were treated with 0.1% DMSO or 100 μM palmostatin B (PalmB) for 18 h. Autophagy was induced by 4 h EBSS treatment or induced and inhibited by 4 h EBSS/2 h treatment with 500 nM BafA1. (A) Representative images of immunoblotting conducted for autophagy markers SQSTM1 and LC3‐II with and without Palm B treatment and autophagy induction. (B) Quantification of immunoblots for SQSTM1 normalized to ACTB/β‐actin. Densitometry was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA. (C) Quantification of immunoblots for LC3‐II normalized to ACTB. Quantification was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA).

Techniques Used: Western Blot, Software

SQSTM1 is palmitoylated and degraded by the lysosome. HeLa cells were induced to undergo autophagy through serum deprivation (−FBS) in the presence or absence of 400 nM BafA1. (A) Palmitoylation of SQSTM1 and Ras was detected by the acyl‐RAC assay. Composite gel, all samples were run on the same gel. (B) During autophagy SQSTM1 palmitoylation increased while (C) total SQSTM1 levels decreased (degraded by the lysosome). BafA1 significantly increased both (B) SQSTM1 palmitoylation and (C) total protein. (D) S‐acylated Ras and (E) protein levels were not significantly affected by either serum deprivation or BafA1 (two‐way ANOVA, n = 3, SEM, * p < 0.05).

Figure Legend Snippet: SQSTM1 is palmitoylated and degraded by the lysosome. HeLa cells were induced to undergo autophagy through serum deprivation (−FBS) in the presence or absence of 400 nM BafA1. (A) Palmitoylation of SQSTM1 and Ras was detected by the acyl‐RAC assay. Composite gel, all samples were run on the same gel. (B) During autophagy SQSTM1 palmitoylation increased while (C) total SQSTM1 levels decreased (degraded by the lysosome). BafA1 significantly increased both (B) SQSTM1 palmitoylation and (C) total protein. (D) S‐acylated Ras and (E) protein levels were not significantly affected by either serum deprivation or BafA1 (two‐way ANOVA, n = 3, SEM, * p < 0.05).

Techniques Used:

SQSTM1 is palmitoylated at the di‐cysteine C289, 290 motif, which is required for localization to the lysosome. (A) IP‐ABE was performed from transfected HeLa cells expressing the indicated forms of SQSTM1‐HA in the presence or absence of BafA1 and palmitoylation was detected by streptavidin (STR). Composite gel, all samples were run on the same gel. (B) Only mutation of the C289, 290 di‐cysteine motif significantly reduced S‐acylation of SQSTM1‐HA, regardless of the addition of BafA1 ( n = 4–7, *< 0.05, ***< 0.0001, two‐way ANOVA). (C) HeLa cells expressing RFP‐LC3 (autophagosome marker) and the indicated SQSTM1‐HA were stained with Lysotracker Far Red for 1‐h prior to fixation. Cells were either grown in complete media (fed) as a control or subjected to growth in serum‐deprived media (fasted) for 4 h to induce autophagy and visualized by confocal microscopy ( n = 3). Scale bar = 10 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (D) SQSTM1‐HA and RFP‐LC3, (E) SQSTM1‐HA and lysosomes, and (F) RFP‐LC3 and lysosomes. Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05. Data are represented as mean ± SEM.

Figure Legend Snippet: SQSTM1 is palmitoylated at the di‐cysteine C289, 290 motif, which is required for localization to the lysosome. (A) IP‐ABE was performed from transfected HeLa cells expressing the indicated forms of SQSTM1‐HA in the presence or absence of BafA1 and palmitoylation was detected by streptavidin (STR). Composite gel, all samples were run on the same gel. (B) Only mutation of the C289, 290 di‐cysteine motif significantly reduced S‐acylation of SQSTM1‐HA, regardless of the addition of BafA1 ( n = 4–7, *< 0.05, ***< 0.0001, two‐way ANOVA). (C) HeLa cells expressing RFP‐LC3 (autophagosome marker) and the indicated SQSTM1‐HA were stained with Lysotracker Far Red for 1‐h prior to fixation. Cells were either grown in complete media (fed) as a control or subjected to growth in serum‐deprived media (fasted) for 4 h to induce autophagy and visualized by confocal microscopy ( n = 3). Scale bar = 10 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (D) SQSTM1‐HA and RFP‐LC3, (E) SQSTM1‐HA and lysosomes, and (F) RFP‐LC3 and lysosomes. Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05. Data are represented as mean ± SEM.

Techniques Used: Transfection, Expressing, Mutagenesis, Marker, Staining, Control, Confocal Microscopy, Comparison

SQSTM1 palmitoylation is significantly reduced in HD. (A, B) SQSTM1 palmitoylation is significantly reduced in the cortex from HD patient brains. (A) Palmitoylation of SQSTM1 was detected in patient brains by acyl‐resin assisted capture (ARAC). Tissues used were from patients of similar age at death, CAG size, and post‐mortem interval. Male and female samples were included in controls and HD samples. Two‐tailed unpaired t ‐test, SEM ( n = 5,4 for Ctrl, HD). Composite gel, all samples were run on the same gel. (B) Total SQSTM1 levels were examined in HD patients. (C, D) Significantly reduced SQSTM1 palmitoylation in the brains of HD mice is restored by fasting. Eight‐month‐old YAC128 mice were fasted for 24 h, as previously described. (C) Palmitoylation of SQSTM1 was detected using IP‐ABE (SQSTM1 is immunoprecipitated prior to ABE—top) and quantified (bottom). The arrow on the blot indicates the antibody heavy chain band. Composite gel, all samples were run on the same gel. (D) Total SQSTM1 levels were immunodetected (top) and quantified (bottom). Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05, *** p < 0.0001. Data are represented as SEM.

Figure Legend Snippet: SQSTM1 palmitoylation is significantly reduced in HD. (A, B) SQSTM1 palmitoylation is significantly reduced in the cortex from HD patient brains. (A) Palmitoylation of SQSTM1 was detected in patient brains by acyl‐resin assisted capture (ARAC). Tissues used were from patients of similar age at death, CAG size, and post‐mortem interval. Male and female samples were included in controls and HD samples. Two‐tailed unpaired t ‐test, SEM ( n = 5,4 for Ctrl, HD). Composite gel, all samples were run on the same gel. (B) Total SQSTM1 levels were examined in HD patients. (C, D) Significantly reduced SQSTM1 palmitoylation in the brains of HD mice is restored by fasting. Eight‐month‐old YAC128 mice were fasted for 24 h, as previously described. (C) Palmitoylation of SQSTM1 was detected using IP‐ABE (SQSTM1 is immunoprecipitated prior to ABE—top) and quantified (bottom). The arrow on the blot indicates the antibody heavy chain band. Composite gel, all samples were run on the same gel. (D) Total SQSTM1 levels were immunodetected (top) and quantified (bottom). Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05, *** p < 0.0001. Data are represented as SEM.

Techniques Used: Two Tailed Test, Immunoprecipitation, Comparison

Palmostatin B increases localization of HTT to the autophagosomes. HeLa cells were treated with 0.1% DMSO or 10 μM Palmostatin B (PalmB) for 16–18 h. (A) Representative images of immunofluorescence conducted on the treated cells for endogenous SQSTM1 and endogenous HTT. Lysosomes were stained with 50 nM LysoT4racker Far Red (Thermofisher Scientific, L12492 ); 1 h incubation at 37°C prior to fixation. Scale bar = 50 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (B) SQSTM1 and HTT, (C) SQSTM1 and lysosomes, and (D) HTT and lysosomes. Statistical significance was determined by the two‐tailed unpaired t ‐test; * p < 0.05, *** p < 0.001. Data are represented as mean ± SEM ( n = 3).

Figure Legend Snippet: Palmostatin B increases localization of HTT to the autophagosomes. HeLa cells were treated with 0.1% DMSO or 10 μM Palmostatin B (PalmB) for 16–18 h. (A) Representative images of immunofluorescence conducted on the treated cells for endogenous SQSTM1 and endogenous HTT. Lysosomes were stained with 50 nM LysoT4racker Far Red (Thermofisher Scientific, L12492 ); 1 h incubation at 37°C prior to fixation. Scale bar = 50 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (B) SQSTM1 and HTT, (C) SQSTM1 and lysosomes, and (D) HTT and lysosomes. Statistical significance was determined by the two‐tailed unpaired t ‐test; * p < 0.05, *** p < 0.001. Data are represented as mean ± SEM ( n = 3).

Techniques Used: Immunofluorescence, Staining, Incubation, Two Tailed Test

Image Search Results

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: Palmostatin B alters autophagy in HeLa cells, assessed through LC3B and SQSTM1 immunoblotting. HeLa cells were treated with 0.1% DMSO or 100 μM palmostatin B (PalmB) for 18 h. Autophagy was induced by 4 h EBSS treatment or induced and inhibited by 4 h EBSS/2 h treatment with 500 nM BafA1. (A) Representative images of immunoblotting conducted for autophagy markers SQSTM1 and LC3‐II with and without Palm B treatment and autophagy induction. (B) Quantification of immunoblots for SQSTM1 normalized to ACTB/β‐actin. Densitometry was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA. (C) Quantification of immunoblots for LC3‐II normalized to ACTB. Quantification was conducted using ImageJ software (mean ± SEM from three independent experiments; two‐way ANOVA).

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Western Blot, Software

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: SQSTM1 is palmitoylated and degraded by the lysosome. HeLa cells were induced to undergo autophagy through serum deprivation (−FBS) in the presence or absence of 400 nM BafA1. (A) Palmitoylation of SQSTM1 and Ras was detected by the acyl‐RAC assay. Composite gel, all samples were run on the same gel. (B) During autophagy SQSTM1 palmitoylation increased while (C) total SQSTM1 levels decreased (degraded by the lysosome). BafA1 significantly increased both (B) SQSTM1 palmitoylation and (C) total protein. (D) S‐acylated Ras and (E) protein levels were not significantly affected by either serum deprivation or BafA1 (two‐way ANOVA, n = 3, SEM, * p < 0.05).

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques:

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: SQSTM1 is palmitoylated at the di‐cysteine C289, 290 motif, which is required for localization to the lysosome. (A) IP‐ABE was performed from transfected HeLa cells expressing the indicated forms of SQSTM1‐HA in the presence or absence of BafA1 and palmitoylation was detected by streptavidin (STR). Composite gel, all samples were run on the same gel. (B) Only mutation of the C289, 290 di‐cysteine motif significantly reduced S‐acylation of SQSTM1‐HA, regardless of the addition of BafA1 ( n = 4–7, *< 0.05, ***< 0.0001, two‐way ANOVA). (C) HeLa cells expressing RFP‐LC3 (autophagosome marker) and the indicated SQSTM1‐HA were stained with Lysotracker Far Red for 1‐h prior to fixation. Cells were either grown in complete media (fed) as a control or subjected to growth in serum‐deprived media (fasted) for 4 h to induce autophagy and visualized by confocal microscopy ( n = 3). Scale bar = 10 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (D) SQSTM1‐HA and RFP‐LC3, (E) SQSTM1‐HA and lysosomes, and (F) RFP‐LC3 and lysosomes. Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Transfection, Expressing, Mutagenesis, Marker, Staining, Control, Confocal Microscopy, Comparison

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: SQSTM1 palmitoylation is significantly reduced in HD. (A, B) SQSTM1 palmitoylation is significantly reduced in the cortex from HD patient brains. (A) Palmitoylation of SQSTM1 was detected in patient brains by acyl‐resin assisted capture (ARAC). Tissues used were from patients of similar age at death, CAG size, and post‐mortem interval. Male and female samples were included in controls and HD samples. Two‐tailed unpaired t ‐test, SEM ( n = 5,4 for Ctrl, HD). Composite gel, all samples were run on the same gel. (B) Total SQSTM1 levels were examined in HD patients. (C, D) Significantly reduced SQSTM1 palmitoylation in the brains of HD mice is restored by fasting. Eight‐month‐old YAC128 mice were fasted for 24 h, as previously described. (C) Palmitoylation of SQSTM1 was detected using IP‐ABE (SQSTM1 is immunoprecipitated prior to ABE—top) and quantified (bottom). The arrow on the blot indicates the antibody heavy chain band. Composite gel, all samples were run on the same gel. (D) Total SQSTM1 levels were immunodetected (top) and quantified (bottom). Statistical significance was determined by two‐way ANOVA with Bonferroni's post hoc comparison; * p < 0.05, *** p < 0.0001. Data are represented as SEM.

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Two Tailed Test, Immunoprecipitation, Comparison

Journal: The FASEB Journal

Article Title: Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy

doi: 10.1096/fj.202401781R

Figure Lengend Snippet: Palmostatin B increases localization of HTT to the autophagosomes. HeLa cells were treated with 0.1% DMSO or 10 μM Palmostatin B (PalmB) for 16–18 h. (A) Representative images of immunofluorescence conducted on the treated cells for endogenous SQSTM1 and endogenous HTT. Lysosomes were stained with 50 nM LysoT4racker Far Red (Thermofisher Scientific, L12492 ); 1 h incubation at 37°C prior to fixation. Scale bar = 50 μm. Co‐localization was measured as Pearson's colocalization coefficient (PCC) for (B) SQSTM1 and HTT, (C) SQSTM1 and lysosomes, and (D) HTT and lysosomes. Statistical significance was determined by the two‐tailed unpaired t ‐test; * p < 0.05, *** p < 0.001. Data are represented as mean ± SEM ( n = 3).

Article Snippet: HA‐tagged SQSTM1 and RFP‐LC3 were gifts from Qing Zhong (Addgene, 28027) [ ] and Tamotsu Yoshimori (Addgene, 21075) [ ], respectively.

Techniques: Immunofluorescence, Staining, Incubation, Two Tailed Test

TGase 2 and HDM2 compete for p53 interaction. TGM2 knockdown increased the interaction of p53 with HDM2, whereas it abolished the interaction with p62 ( a and b ). ACHN and CAKI-1 cells were transfected with siRNA for TGM2 ( a ) or HDM2 ( b ) for 48 h under starvation conditions. Whole-cell extracts (left) or p53 immunoprecipitates (right) were subjected to immunoblotting for TGase 2, HDM2, p53 and p62. ( c ) The induction of DNA damage inhibited the binding of TGase 2 to p53 and induced p53 phosphorylation. CAKI-1 and ACHN cells were treated with doxorubicin (1 μ M) or etoposide (50 μ M) for 24 h; whole-cell extracts were subjected to p53 immunoprecipitation and western blotting. ( d ) The activated p53 mimic (S15E) did not interact with TGase 2. CAKI-1 and ACHN were transfected with expression plasmids for p53 (wild type), p53 (S15E), or p53 (S15A) for 24 h, and the cell lysates were immunoprecipitated with a His-tag antibody and subjected to immunoblotting. ( e ) p53 stabilization (p-p53) by doxorubicin blocked interaction with TGase 2 in a time-dependent manner. After doxorubicin (1 μ M) treatment for up to 20 h, cell lysates were immunoprecipitated with an anti-p53 antibody and subjected to immunoblotting. The blots are representative of three independent experiments

Journal: Cell Death & Disease

Article Title: Renal cell carcinoma escapes death by p53 depletion through transglutaminase 2-chaperoned autophagy

doi: 10.1038/cddis.2016.14

Figure Lengend Snippet: TGase 2 and HDM2 compete for p53 interaction. TGM2 knockdown increased the interaction of p53 with HDM2, whereas it abolished the interaction with p62 ( a and b ). ACHN and CAKI-1 cells were transfected with siRNA for TGM2 ( a ) or HDM2 ( b ) for 48 h under starvation conditions. Whole-cell extracts (left) or p53 immunoprecipitates (right) were subjected to immunoblotting for TGase 2, HDM2, p53 and p62. ( c ) The induction of DNA damage inhibited the binding of TGase 2 to p53 and induced p53 phosphorylation. CAKI-1 and ACHN cells were treated with doxorubicin (1 μ M) or etoposide (50 μ M) for 24 h; whole-cell extracts were subjected to p53 immunoprecipitation and western blotting. ( d ) The activated p53 mimic (S15E) did not interact with TGase 2. CAKI-1 and ACHN were transfected with expression plasmids for p53 (wild type), p53 (S15E), or p53 (S15A) for 24 h, and the cell lysates were immunoprecipitated with a His-tag antibody and subjected to immunoblotting. ( e ) p53 stabilization (p-p53) by doxorubicin blocked interaction with TGase 2 in a time-dependent manner. After doxorubicin (1 μ M) treatment for up to 20 h, cell lysates were immunoprecipitated with an anti-p53 antibody and subjected to immunoblotting. The blots are representative of three independent experiments

Article Snippet: The TGase 2 antibody from Lab Vision (clone CUB 7402; Fremont, CA, USA), the β -actin (SC-47778), HDM2 (SC-813), His (SC-803) and p53 (SC-126) and p62/SQSTM1 (SC-SC-25575), HA (SC-7392, SC-805) antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and p-p53(ser15) (#9284 S) LC3B (#3868) antibodies from Cell Signaling Technologies (Beverly, MA, USA) and FLAG(F1804, F7425) antibody from Sigma-Aldrich (St. Louis, MI, USA).

Techniques: Knockdown, Transfection, Western Blot, Binding Assay, Phospho-proteomics, Immunoprecipitation, Expressing

TGase 2 chaperones p53 to p62. ( a and b ) TGase 2 knockdown abolished the interaction of p53 to p62 as well as the interaction of TGase 2 to p53 and p62. TGM2 was silenced in ACHN ( a ) or CAKI-1 ( b ) cells for 48 h under starvation conditions, and then cell extracts were subjected to immunoprecipitation of TGase 2, p53, and p62. ( c ) TGase 2 activity is not required for interacting with p53. Wild-type or catalytically inactive TGase 2 (double mutant, C277S and C370A) was co-transfected with p62 into HEK293 cells. TGase 2 was immunoprecipitated using an anti-HA-tag antibody, followed by immunoblotting of TGase 2, p53 and p62

Journal: Cell Death & Disease

Article Title: Renal cell carcinoma escapes death by p53 depletion through transglutaminase 2-chaperoned autophagy

doi: 10.1038/cddis.2016.14

Figure Lengend Snippet: TGase 2 chaperones p53 to p62. ( a and b ) TGase 2 knockdown abolished the interaction of p53 to p62 as well as the interaction of TGase 2 to p53 and p62. TGM2 was silenced in ACHN ( a ) or CAKI-1 ( b ) cells for 48 h under starvation conditions, and then cell extracts were subjected to immunoprecipitation of TGase 2, p53, and p62. ( c ) TGase 2 activity is not required for interacting with p53. Wild-type or catalytically inactive TGase 2 (double mutant, C277S and C370A) was co-transfected with p62 into HEK293 cells. TGase 2 was immunoprecipitated using an anti-HA-tag antibody, followed by immunoblotting of TGase 2, p53 and p62

Techniques: Knockdown, Immunoprecipitation, Activity Assay, Mutagenesis, Transfection, Western Blot

TGase 2 makes a trimer complex with p53 and p62. ( a ) The N terminus of p62 (residues 85–110) is the TGase 2-binding site. HEK293 cells were transfected with plasmids encoding HA-tagged TGase 2 (HA-TG2) and either wild-type p62 (p3xFLAG p62 wild type) or a p62 deletion mutant. The proteins were co-immunoprecipitated from cell extracts using an anti-HA-tag antibody and subjected to immunoblotting. ( b ) The C terminus of TGase 2 is the p62-binding domain ( β -barrel domains, residues 461–687). HEK293 cells were transfected with p62 (p3XFLAG p62 wild type) and either wild-type TGase 2 (HA-TG2) or a TGase 2 deletion mutant. The proteins were co-immunoprecipitated from cell extracts using an anti-HA-tag antibody and subjected to immunoblotting. ( c ) The N terminus of TGase 2 is the p53-binding domain (residues 1–139). HEK293 cells were transfected with wild-type p53 (p3xFLAG p53) and either HA-tagged wild-type TGase 2 (HA-TG2) or a deletion mutant of TGase 2. Proteins were immunoprecipitated using an anti-HA-tag antibody. ( d ) Primary structures of p62, p53 and TGase 2 proteins. CRD, C-terminal regulatory domain, lysine rich domain; DBD, DNA-binding domain; KIR, Keap1-interacting region; LIR, LC3-interacting region; p53, tumor suppressor; p62, sequestosome 1; TAD, transcription-activation domain or transactivation domain; TBD, TRAF6-binding domain; TET, tetramerization domain; ZZ, zinc-finger domain

Journal: Cell Death & Disease

Article Title: Renal cell carcinoma escapes death by p53 depletion through transglutaminase 2-chaperoned autophagy

doi: 10.1038/cddis.2016.14

Figure Lengend Snippet: TGase 2 makes a trimer complex with p53 and p62. ( a ) The N terminus of p62 (residues 85–110) is the TGase 2-binding site. HEK293 cells were transfected with plasmids encoding HA-tagged TGase 2 (HA-TG2) and either wild-type p62 (p3xFLAG p62 wild type) or a p62 deletion mutant. The proteins were co-immunoprecipitated from cell extracts using an anti-HA-tag antibody and subjected to immunoblotting. ( b ) The C terminus of TGase 2 is the p62-binding domain ( β -barrel domains, residues 461–687). HEK293 cells were transfected with p62 (p3XFLAG p62 wild type) and either wild-type TGase 2 (HA-TG2) or a TGase 2 deletion mutant. The proteins were co-immunoprecipitated from cell extracts using an anti-HA-tag antibody and subjected to immunoblotting. ( c ) The N terminus of TGase 2 is the p53-binding domain (residues 1–139). HEK293 cells were transfected with wild-type p53 (p3xFLAG p53) and either HA-tagged wild-type TGase 2 (HA-TG2) or a deletion mutant of TGase 2. Proteins were immunoprecipitated using an anti-HA-tag antibody. ( d ) Primary structures of p62, p53 and TGase 2 proteins. CRD, C-terminal regulatory domain, lysine rich domain; DBD, DNA-binding domain; KIR, Keap1-interacting region; LIR, LC3-interacting region; p53, tumor suppressor; p62, sequestosome 1; TAD, transcription-activation domain or transactivation domain; TBD, TRAF6-binding domain; TET, tetramerization domain; ZZ, zinc-finger domain

Techniques: Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Activation Assay

Doxorubicin amplifies the therapeutic response of RCC to the TGase 2 inhibitor GK921. ( a ) ACHN-luciferase cells were injected subcutaneously into 6–8-week-old female BALB/c nude mice. When the tumor volume reached 100 mm 3 , mice were treated orally, 5 days/week, with GK921 (2 mg/kg/day), doxorubicin (1 mg/kg/day), both or vehicle ( n =3 per each group). After 5 weeks of treatment, tumor size was measured by the photon flux from luciferin and expressed on a color scale and as photons/s/cm 2 /steradian (left). Tumor volume was also measured using calipers (right). ( b – e ) Bioinformatics analysis of p62 gene ( SQSTM1 ) and TGase 2 gene ( TGM2 ) expression in clear cell RCC patients. ( b ) Lower p62/ SQSTM1 expression in normal kidney samples than in matched clear cell RCC samples ( P =3.1367 × 10 −12 ). ( c ) Lower TGM2 expression in normal kidney samples than in matched clear cell RCC samples ( P =3.5708 × 10 −15 ). ( d ) Patients with high levels of p62/ SQSTM1 expression (red) had lower overall survival (OS) than those with low levels (blue). Log-rank test, P =0.00618. ( e ) Patients with high levels of expression of TGM2 (red) also had a reduced OS. Gene expressions are colored red; higher levels of gene expression cut off z -score>2 σ . Log-rank test, P =0.00579

Journal: Cell Death & Disease

Article Title: Renal cell carcinoma escapes death by p53 depletion through transglutaminase 2-chaperoned autophagy

doi: 10.1038/cddis.2016.14

Figure Lengend Snippet: Doxorubicin amplifies the therapeutic response of RCC to the TGase 2 inhibitor GK921. ( a ) ACHN-luciferase cells were injected subcutaneously into 6–8-week-old female BALB/c nude mice. When the tumor volume reached 100 mm 3 , mice were treated orally, 5 days/week, with GK921 (2 mg/kg/day), doxorubicin (1 mg/kg/day), both or vehicle ( n =3 per each group). After 5 weeks of treatment, tumor size was measured by the photon flux from luciferin and expressed on a color scale and as photons/s/cm 2 /steradian (left). Tumor volume was also measured using calipers (right). ( b – e ) Bioinformatics analysis of p62 gene ( SQSTM1 ) and TGase 2 gene ( TGM2 ) expression in clear cell RCC patients. ( b ) Lower p62/ SQSTM1 expression in normal kidney samples than in matched clear cell RCC samples ( P =3.1367 × 10 −12 ). ( c ) Lower TGM2 expression in normal kidney samples than in matched clear cell RCC samples ( P =3.5708 × 10 −15 ). ( d ) Patients with high levels of p62/ SQSTM1 expression (red) had lower overall survival (OS) than those with low levels (blue). Log-rank test, P =0.00618. ( e ) Patients with high levels of expression of TGM2 (red) also had a reduced OS. Gene expressions are colored red; higher levels of gene expression cut off z -score>2 σ . Log-rank test, P =0.00579

Techniques: Clinical Proteomics, Luciferase, Injection, Expressing, Gene Expression

Model of the interactions of TGase 2, p53 and p62 based on the experimental data. The N terminus of TGase 2 interacts with the N terminus of p53, and the C terminus of TGase 2 interacts with the N terminus of p62 simultaneously. The C terminus of p62 in the complex is free and transfers the complex to LC3 in the phagophore. P53 is polymerized in the autophagosome when calcium increases. Later, the autophagosome and lysosome fuse into an autolysosome, which degrades all crosslinked materials. The TGase 2-binding site of p53 was identified as the HDM2-binding site in . Previously, we found that the TGase 2-targeting site is the DBD domain. CD, complexing domain; DBD, DNA-binding domain

Journal: Cell Death & Disease

Article Title: Renal cell carcinoma escapes death by p53 depletion through transglutaminase 2-chaperoned autophagy

doi: 10.1038/cddis.2016.14

Figure Lengend Snippet: Model of the interactions of TGase 2, p53 and p62 based on the experimental data. The N terminus of TGase 2 interacts with the N terminus of p53, and the C terminus of TGase 2 interacts with the N terminus of p62 simultaneously. The C terminus of p62 in the complex is free and transfers the complex to LC3 in the phagophore. P53 is polymerized in the autophagosome when calcium increases. Later, the autophagosome and lysosome fuse into an autolysosome, which degrades all crosslinked materials. The TGase 2-binding site of p53 was identified as the HDM2-binding site in . Previously, we found that the TGase 2-targeting site is the DBD domain. CD, complexing domain; DBD, DNA-binding domain

Techniques: Binding Assay

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1) Product Images from "Reduced Palmitoylation of SQSTM1 /p62 in Huntington Disease Is Associated With Impaired Autophagy"

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