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Addgene inc pgex 6p 1 p37 shpmut

( A ) Domain structure of human p47 and <t>p37.</t> ( B and C ) Protein levels in HeLa cells treated with control or p47 siRNA (B) or control or p47 KO cells treated with cycloheximide (CHX) for 6 hours with 10 μM MG132 where indicated (C); n = 3, two-tailed paired Student’s t test. ( D ) Schematic representation of constructs expressing p47 mutant protein. ( E ) Immunoprecipitation of a WT and mutant p47-FLAG from HeLa cells expressing p37-Clover; n = 4. ( F ) In vitro binding assay. Recombinant p47-GST was immunoprecipitated together with purified WT p37, VCP nonbinding p37 SHP mutant, and VCP; n = 3, one-sample t test and two-tailed paired Student’s t test. ( G and H ) p47 WT and p47 mutants were expressed for 24 hours in p47 KO HeLa cells. (G) Western blot analysis; n = 4, one-way ANOVA ( P = 0.0012) with post hoc Tukey test, p37 levels were normalized to p47-FLAG. (H) Immunocytochemistry for LC3 and FLAG after treatment with 400 nM BafA1 for 4 hours; n = 3, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( I ) Protein levels in HeLa cells treated with control or p47 siRNA for 48 hours and p37-FLAG expression for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours where indicated; n = 4, one-sample t test and two-tailed paired Student’s t test. ( J ) p47 KO HeLa cells were treated with p37 siRNA for 48 hours and subsequent expression of p47-FLAG for 24 hours, followed by 400 nM BafA1 4-hour treatment and immunostaining for LC3 and FLAG; n = 3 (number of counted cells >50 per condition), one-way ANOVA ( P = 0.0035) with post hoc Tukey test. Data are means ± SEM. Scale bars, 10 μm. *, unspecific band. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Pgex 6p 1 P37 Shpmut, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 2 article reviews

pgex 6p 1 p37 shpmut - by Bioz Stars, 2026-08

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1) Product Images from "p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol"

Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

Journal: Science Advances

doi: 10.1126/sciadv.adl6082

( A ) Domain structure of human p47 and p37. ( B and C ) Protein levels in HeLa cells treated with control or p47 siRNA (B) or control or p47 KO cells treated with cycloheximide (CHX) for 6 hours with 10 μM MG132 where indicated (C); n = 3, two-tailed paired Student’s t test. ( D ) Schematic representation of constructs expressing p47 mutant protein. ( E ) Immunoprecipitation of a WT and mutant p47-FLAG from HeLa cells expressing p37-Clover; n = 4. ( F ) In vitro binding assay. Recombinant p47-GST was immunoprecipitated together with purified WT p37, VCP nonbinding p37 SHP mutant, and VCP; n = 3, one-sample t test and two-tailed paired Student’s t test. ( G and H ) p47 WT and p47 mutants were expressed for 24 hours in p47 KO HeLa cells. (G) Western blot analysis; n = 4, one-way ANOVA ( P = 0.0012) with post hoc Tukey test, p37 levels were normalized to p47-FLAG. (H) Immunocytochemistry for LC3 and FLAG after treatment with 400 nM BafA1 for 4 hours; n = 3, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( I ) Protein levels in HeLa cells treated with control or p47 siRNA for 48 hours and p37-FLAG expression for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours where indicated; n = 4, one-sample t test and two-tailed paired Student’s t test. ( J ) p47 KO HeLa cells were treated with p37 siRNA for 48 hours and subsequent expression of p47-FLAG for 24 hours, followed by 400 nM BafA1 4-hour treatment and immunostaining for LC3 and FLAG; n = 3 (number of counted cells >50 per condition), one-way ANOVA ( P = 0.0035) with post hoc Tukey test. Data are means ± SEM. Scale bars, 10 μm. *, unspecific band. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Figure Legend Snippet: ( A ) Domain structure of human p47 and p37. ( B and C ) Protein levels in HeLa cells treated with control or p47 siRNA (B) or control or p47 KO cells treated with cycloheximide (CHX) for 6 hours with 10 μM MG132 where indicated (C); n = 3, two-tailed paired Student’s t test. ( D ) Schematic representation of constructs expressing p47 mutant protein. ( E ) Immunoprecipitation of a WT and mutant p47-FLAG from HeLa cells expressing p37-Clover; n = 4. ( F ) In vitro binding assay. Recombinant p47-GST was immunoprecipitated together with purified WT p37, VCP nonbinding p37 SHP mutant, and VCP; n = 3, one-sample t test and two-tailed paired Student’s t test. ( G and H ) p47 WT and p47 mutants were expressed for 24 hours in p47 KO HeLa cells. (G) Western blot analysis; n = 4, one-way ANOVA ( P = 0.0012) with post hoc Tukey test, p37 levels were normalized to p47-FLAG. (H) Immunocytochemistry for LC3 and FLAG after treatment with 400 nM BafA1 for 4 hours; n = 3, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( I ) Protein levels in HeLa cells treated with control or p47 siRNA for 48 hours and p37-FLAG expression for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours where indicated; n = 4, one-sample t test and two-tailed paired Student’s t test. ( J ) p47 KO HeLa cells were treated with p37 siRNA for 48 hours and subsequent expression of p47-FLAG for 24 hours, followed by 400 nM BafA1 4-hour treatment and immunostaining for LC3 and FLAG; n = 3 (number of counted cells >50 per condition), one-way ANOVA ( P = 0.0035) with post hoc Tukey test. Data are means ± SEM. Scale bars, 10 μm. *, unspecific band. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques Used: Control, Two Tailed Test, Construct, Expressing, Mutagenesis, Immunoprecipitation, In Vitro, Binding Assay, Recombinant, Purification, Western Blot, Immunocytochemistry, Immunostaining, Transfection, Over Expression, Knockdown

( A ) Control, p37 KO, and p37 KO HeLa cells reconstituted with p37-FLAG were treated with 400 nM BafA1 for 4 hours, followed by immunostaining for LC3 and FLAG; n = 3, one-way ANOVA ( P = 0.004) with post hoc Tukey test. ( B ) iNeurons treated with lentiviral-delivered p37 shRNA (#81 or #83) for 4 days were treated with 400 nM BafA1 for 6 hours; n = 4, one-sample t test. ( C ) Control, p37 KO, and p37 KO with p37-FLAG–expressing HeLa cells were incubated in EBSS for 2 hours, followed by immunostaining for PI(3)P; n = 3 to 4, one-sample t test and two-tailed unpaired Student’s t test. ( D and E ) Endogenous ATG14L was immunoprecipitated from control and p37 KO HeLa cells (D) or control and p37-Clover–overexpressing HeLa cells (E); n = 4 to 5; one-sample t test. ( F to H ) Control or p37-FLAG–overexpressing cells were incubated in EBSS for 2 hours (F and G) and treated with 5 μM CB-5083 for 3 hours (G) or 400 nM BafA1 for 4 hours (H) where indicated, followed by immunostaining for PI(3)P (F), WIPI2 (G), or LC3 (H); n = 3, two-tailed unpaired Student’s t test. ( I ) HeLa cells expressing WT or SHP mutant p37-FLAG for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours; n = 3 to 4, one-sample t test. ( J ) Control or Beclin-1 KO HeLa cells expressing p37-FLAG were treated with 400 nM BafA1 for 4 hours; n = 5, one-sample t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Figure Legend Snippet: ( A ) Control, p37 KO, and p37 KO HeLa cells reconstituted with p37-FLAG were treated with 400 nM BafA1 for 4 hours, followed by immunostaining for LC3 and FLAG; n = 3, one-way ANOVA ( P = 0.004) with post hoc Tukey test. ( B ) iNeurons treated with lentiviral-delivered p37 shRNA (#81 or #83) for 4 days were treated with 400 nM BafA1 for 6 hours; n = 4, one-sample t test. ( C ) Control, p37 KO, and p37 KO with p37-FLAG–expressing HeLa cells were incubated in EBSS for 2 hours, followed by immunostaining for PI(3)P; n = 3 to 4, one-sample t test and two-tailed unpaired Student’s t test. ( D and E ) Endogenous ATG14L was immunoprecipitated from control and p37 KO HeLa cells (D) or control and p37-Clover–overexpressing HeLa cells (E); n = 4 to 5; one-sample t test. ( F to H ) Control or p37-FLAG–overexpressing cells were incubated in EBSS for 2 hours (F and G) and treated with 5 μM CB-5083 for 3 hours (G) or 400 nM BafA1 for 4 hours (H) where indicated, followed by immunostaining for PI(3)P (F), WIPI2 (G), or LC3 (H); n = 3, two-tailed unpaired Student’s t test. ( I ) HeLa cells expressing WT or SHP mutant p37-FLAG for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours; n = 3 to 4, one-sample t test. ( J ) Control or Beclin-1 KO HeLa cells expressing p37-FLAG were treated with 400 nM BafA1 for 4 hours; n = 5, one-sample t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques Used: Control, Immunostaining, shRNA, Expressing, Incubation, Two Tailed Test, Immunoprecipitation, Mutagenesis, Transfection, Over Expression, Construct, Knockdown

( A ) Mouse striatal cells with WT (Q7/Q7) or mutant (Q111/Q111) huntingtin overexpressing p37-FLAG were analyzed by western blotting; n = 3, one-sample t test. ( B ) HTTQ74-EGFP aggregates in control and p37-overexpressing cells in control or ATG16 KO HeLa cells; n = 3, two-tailed paired Student’s t test. ( C and D ) A53T-SNCA-EGFP HeLa cells overexpressing p37-FLAG, p47-FLAG, or VCP-HA (C) or overexpressing WT or SHP mutant p37 (D) were analyzed by FACS; n = 5, one-sample t test. ( E ) Control, p37 KO, and p37 KO expressing p37-FLAG HeLa cells were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 3, one-way ANOVA ( P = 0.0074) with post hoc Tukey test. ( F ) A53T-SNCA-EGFP HeLa cells were treated with control or p37 siRNA for 48 hours, followed by FACS analysis; n = 3, one-sample t test. ( G ) Control and ATG16 KO HeLa cells overexpressing p37-FLAG were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 4, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( H ) Control and p37-FLAG–overexpressing HeLa cells pre-treated with 5 μM CB-5083 or DMSO for 1 hour were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 3, two-tailed paired Student’s t test. ( I ) Quantification of total, cytosolic, and nuclear Ub + inclusions in control and p37-overexpressing cells; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. Scale bars, 10 μm. *, unspecific band; SE, short exposure. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Figure Legend Snippet: ( A ) Mouse striatal cells with WT (Q7/Q7) or mutant (Q111/Q111) huntingtin overexpressing p37-FLAG were analyzed by western blotting; n = 3, one-sample t test. ( B ) HTTQ74-EGFP aggregates in control and p37-overexpressing cells in control or ATG16 KO HeLa cells; n = 3, two-tailed paired Student’s t test. ( C and D ) A53T-SNCA-EGFP HeLa cells overexpressing p37-FLAG, p47-FLAG, or VCP-HA (C) or overexpressing WT or SHP mutant p37 (D) were analyzed by FACS; n = 5, one-sample t test. ( E ) Control, p37 KO, and p37 KO expressing p37-FLAG HeLa cells were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 3, one-way ANOVA ( P = 0.0074) with post hoc Tukey test. ( F ) A53T-SNCA-EGFP HeLa cells were treated with control or p37 siRNA for 48 hours, followed by FACS analysis; n = 3, one-sample t test. ( G ) Control and ATG16 KO HeLa cells overexpressing p37-FLAG were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 4, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( H ) Control and p37-FLAG–overexpressing HeLa cells pre-treated with 5 μM CB-5083 or DMSO for 1 hour were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 3, two-tailed paired Student’s t test. ( I ) Quantification of total, cytosolic, and nuclear Ub + inclusions in control and p37-overexpressing cells; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. Scale bars, 10 μm. *, unspecific band; SE, short exposure. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques Used: Mutagenesis, Western Blot, Control, Two Tailed Test, Expressing, Immunostaining, Ubiquitin Proteomics, Transfection, Over Expression, Construct, Knockdown

$( A ) Control, p37 KO, p37 KO expressing WT p37-FLAG, and p37 KO expressing WT p47-FLAG HeLa cells were immunostained for VCP and FLAG; n = 3, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( B ) Cytosolic and nuclear fractions from control, p37 KO, and p37 KO expressing WT or SHP mutant p37-FLAG HeLa cells were analyzed for VCP protein levels with Lamin B1 as a nuclear marker and GAPDH as a cytosolic marker; n = 4, one-sample t test and two-tailed paired Student’s t test (for nuclear p37 KO analysis). ( C and D ) iNeurons treated with lentiviral-delivered shRNA#81 against p37 (C) or expressing lentiviral-delivered p37-Clover (D) for 4 days were immunostained for VCP and p37; n = 3, two-tailed paired Student’s t test. ( E ) HeLa cells incubated in EBSS for 6 hours were immunostained for VCP and analyzed for VCP signal in cytosol and nucleus; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.$
Figure Legend Snippet: ( A ) Control, p37 KO, p37 KO expressing WT p37-FLAG, and p37 KO expressing WT p47-FLAG HeLa cells were immunostained for VCP and FLAG; n = 3, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( B ) Cytosolic and nuclear fractions from control, p37 KO, and p37 KO expressing WT or SHP mutant p37-FLAG HeLa cells were analyzed for VCP protein levels with Lamin B1 as a nuclear marker and GAPDH as a cytosolic marker; n = 4, one-sample t test and two-tailed paired Student’s t test (for nuclear p37 KO analysis). ( C and D ) iNeurons treated with lentiviral-delivered shRNA#81 against p37 (C) or expressing lentiviral-delivered p37-Clover (D) for 4 days were immunostained for VCP and p37; n = 3, two-tailed paired Student’s t test. ( E ) HeLa cells incubated in EBSS for 6 hours were immunostained for VCP and analyzed for VCP signal in cytosol and nucleus; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques Used: Control, Expressing, Mutagenesis, Marker, Two Tailed Test, shRNA, Incubation, Transfection, Over Expression, Construct, Knockdown

$( A ) HeLa cells expressing WT or SHP mutant p37-FLAG were immunostained for ubiquitin; n = 3, one-way ANOVA ( P = 0.0001) with post hoc Tukey test. ( B ) Control and p37-FLAG–overexpressing HeLa cells were treated with CHX, followed by isolation of the nuclear fraction, n = 3, two-tailed paired Student’s t test. ( C ) Nucleoplasm and chromatin fraction isolated from control and p37 KO HeLa cells. ( D ) Control and p37 KO HeLa cells were treated with CHX, followed by isolation of the nuclear fraction, n = 3, two-tailed paired Student’s t test. ( E and F ) Control, p37 KO, and p37 KO HeLa cells expressing either WT or SHP mutant p37-FLAG were treated with mitomycin C (1 μg/ml) for 2 hours, followed by immunostaining; statistical analysis in (F); n = 3, one-way ANOVA ( P = 0.0049) with post hoc Tukey test. ( G and H ) iNeurons treated with lentiviral-delivered shRNA#83 against p37 (G) or expressing lentiviral-delivered p37-Clover (H) were treated with mitomycin C (1 μg/ml) for 6 hours, followed by immunostaining; two-tailed paired Student’s t test; n = 5 for (G); n = 3 for (H). ( I and J ) Control and p37 KO HeLa cells treated with mitomycin C (1 μg/ml) for 2 hours in the presence or absence of 5 μM CB-5083 were immunostained for 53BP1; quantification of the total area of 53BP1 foci in (J); n = 3, two-tailed paired Student’s t test. ( K ) Control and p37 KO cells were treated with mitomycin C (1 or 2 μg/ml) for 32 hours, and cell death was monitored every 4 hours; data are represented as slope values of cell death curve over time; n = 4 to 5, one-way ANOVA ( P = 0.0091) with post hoc Tukey test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments we used nontargeting control siRNAs.$
Figure Legend Snippet: ( A ) HeLa cells expressing WT or SHP mutant p37-FLAG were immunostained for ubiquitin; n = 3, one-way ANOVA ( P = 0.0001) with post hoc Tukey test. ( B ) Control and p37-FLAG–overexpressing HeLa cells were treated with CHX, followed by isolation of the nuclear fraction, n = 3, two-tailed paired Student’s t test. ( C ) Nucleoplasm and chromatin fraction isolated from control and p37 KO HeLa cells. ( D ) Control and p37 KO HeLa cells were treated with CHX, followed by isolation of the nuclear fraction, n = 3, two-tailed paired Student’s t test. ( E and F ) Control, p37 KO, and p37 KO HeLa cells expressing either WT or SHP mutant p37-FLAG were treated with mitomycin C (1 μg/ml) for 2 hours, followed by immunostaining; statistical analysis in (F); n = 3, one-way ANOVA ( P = 0.0049) with post hoc Tukey test. ( G and H ) iNeurons treated with lentiviral-delivered shRNA#83 against p37 (G) or expressing lentiviral-delivered p37-Clover (H) were treated with mitomycin C (1 μg/ml) for 6 hours, followed by immunostaining; two-tailed paired Student’s t test; n = 5 for (G); n = 3 for (H). ( I and J ) Control and p37 KO HeLa cells treated with mitomycin C (1 μg/ml) for 2 hours in the presence or absence of 5 μM CB-5083 were immunostained for 53BP1; quantification of the total area of 53BP1 foci in (J); n = 3, two-tailed paired Student’s t test. ( K ) Control and p37 KO cells were treated with mitomycin C (1 or 2 μg/ml) for 32 hours, and cell death was monitored every 4 hours; data are represented as slope values of cell death curve over time; n = 4 to 5, one-way ANOVA ( P = 0.0091) with post hoc Tukey test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments we used nontargeting control siRNAs.

Techniques Used: Expressing, Mutagenesis, Ubiquitin Proteomics, Control, Isolation, Two Tailed Test, Immunostaining, shRNA, Transfection, Over Expression, Construct, Knockdown

$( A and B ) Control, heterozygous VCP R159H mutant (WT/R159H), homozygous VCP R159H mutant (R159H/R159H), and control revertant (Rev) iNeurons were immunostained for VCP (A) or cytosolic and nuclear fractions were isolated (B); quantification of nuclear VCP signal in (A), n = 3, two-tailed paired Student’s t test; n = 3 to 5 in (B), one-sample t test. ( C ) Control, heterozygous VCP R159H, and homozygous VCP R159H mutant iNeurons were treated with mitomycin C (1 μg/ml) for 60 hours to measure cell death; n = 4; one-sample t test. ( D ) Endogenous immunoprecipitation of VCP from control, heterozygous VCP R159H, homozygous VCP R159H mutant, and control revertant iNeurons. ( E and F ) Control, heterozygous VCP R159H, and homozygous VCP R159H mutant iNeurons were treated with lentiviral-delivered control or shRNA against p37 for 4 days and immunostained; quantification of nuclear VCP signal in (E), n = 3; quantification of γ-H2AX-Ser 319 intensity in (F), n = 4, two-tailed paired Student’s t test. ( G ) p37 coordinates the shuttling and local functions of VCP between the cytosol and nucleus. p47 binding to p37 prevents p37 proteasomal degradation. An increase in p37 levels leads to increased VCP localization in the cytosol, resulting in its enhanced function in ERAD and autophagy, but impaired function in CAD and DNA damage repair. Depletion of p37 promotes VCP nuclear localization and impairs VCP function in ERAD and autophagy but enhances its function in CAD and DNA damage repair. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.$
Figure Legend Snippet: ( A and B ) Control, heterozygous VCP R159H mutant (WT/R159H), homozygous VCP R159H mutant (R159H/R159H), and control revertant (Rev) iNeurons were immunostained for VCP (A) or cytosolic and nuclear fractions were isolated (B); quantification of nuclear VCP signal in (A), n = 3, two-tailed paired Student’s t test; n = 3 to 5 in (B), one-sample t test. ( C ) Control, heterozygous VCP R159H, and homozygous VCP R159H mutant iNeurons were treated with mitomycin C (1 μg/ml) for 60 hours to measure cell death; n = 4; one-sample t test. ( D ) Endogenous immunoprecipitation of VCP from control, heterozygous VCP R159H, homozygous VCP R159H mutant, and control revertant iNeurons. ( E and F ) Control, heterozygous VCP R159H, and homozygous VCP R159H mutant iNeurons were treated with lentiviral-delivered control or shRNA against p37 for 4 days and immunostained; quantification of nuclear VCP signal in (E), n = 3; quantification of γ-H2AX-Ser 319 intensity in (F), n = 4, two-tailed paired Student’s t test. ( G ) p37 coordinates the shuttling and local functions of VCP between the cytosol and nucleus. p47 binding to p37 prevents p37 proteasomal degradation. An increase in p37 levels leads to increased VCP localization in the cytosol, resulting in its enhanced function in ERAD and autophagy, but impaired function in CAD and DNA damage repair. Depletion of p37 promotes VCP nuclear localization and impairs VCP function in ERAD and autophagy but enhances its function in CAD and DNA damage repair. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques Used: Control, Mutagenesis, Isolation, Two Tailed Test, Immunoprecipitation, shRNA, Binding Assay, Transfection, Over Expression, Construct, Knockdown

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Journal: Science Advances

Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

doi: 10.1126/sciadv.adl6082

Figure Lengend Snippet: ( A ) Domain structure of human p47 and p37. ( B and C ) Protein levels in HeLa cells treated with control or p47 siRNA (B) or control or p47 KO cells treated with cycloheximide (CHX) for 6 hours with 10 μM MG132 where indicated (C); n = 3, two-tailed paired Student’s t test. ( D ) Schematic representation of constructs expressing p47 mutant protein. ( E ) Immunoprecipitation of a WT and mutant p47-FLAG from HeLa cells expressing p37-Clover; n = 4. ( F ) In vitro binding assay. Recombinant p47-GST was immunoprecipitated together with purified WT p37, VCP nonbinding p37 SHP mutant, and VCP; n = 3, one-sample t test and two-tailed paired Student’s t test. ( G and H ) p47 WT and p47 mutants were expressed for 24 hours in p47 KO HeLa cells. (G) Western blot analysis; n = 4, one-way ANOVA ( P = 0.0012) with post hoc Tukey test, p37 levels were normalized to p47-FLAG. (H) Immunocytochemistry for LC3 and FLAG after treatment with 400 nM BafA1 for 4 hours; n = 3, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( I ) Protein levels in HeLa cells treated with control or p47 siRNA for 48 hours and p37-FLAG expression for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours where indicated; n = 4, one-sample t test and two-tailed paired Student’s t test. ( J ) p47 KO HeLa cells were treated with p37 siRNA for 48 hours and subsequent expression of p47-FLAG for 24 hours, followed by 400 nM BafA1 4-hour treatment and immunostaining for LC3 and FLAG; n = 3 (number of counted cells >50 per condition), one-way ANOVA ( P = 0.0035) with post hoc Tukey test. Data are means ± SEM. Scale bars, 10 μm. *, unspecific band. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Article Snippet: The following DNA constructs were used in this study: VCP(wt)-EGFP was a gift from N. Dantuma (Addgene, plasmid #23971; RRID:Addgene_23971); VCP(R155H)-EGFP was a gift from N. Dantuma (Addgene, plasmid #23972; RRID:Addgene_23972); pcDNA5FRT/TO-p37-Strep-HA was a gift from H. Meyer (Addgene, plasmid #113485; RRID:Addgene_113485); pGEX-6P-1 p37 was a gift from H. Meyer (Addgene, plasmid #113500; RRID:Addgene_113500); pGEX-6P-1 p37 SHPmut was a gift from H. Meyer (Addgene, plasmid #113503; RRID:Addgene_113503); pCW57.1 was a gift from D. Root (Addgene, plasmid #41393; RRID:Addgene_41393). pGEX-VCP-GST was shared by R. Schröder and C. Clemen (University Hospital Erlangen, Erlangen, Germany). pRK5-FLAG vector, pRK5-FLAG-p47WT, pRK5-FLAG-p47∆UBA, pRK5-FLAG-p47∆UBX, and pRK5-FLAG-p47V15A/L34A/Y42A were shared by J. Inoue ( ) (Institute of Medical Science, University of Tokyo).

Techniques: Control, Two Tailed Test, Construct, Expressing, Mutagenesis, Immunoprecipitation, In Vitro, Binding Assay, Recombinant, Purification, Western Blot, Immunocytochemistry, Immunostaining, Transfection, Over Expression, Knockdown

Journal: Science Advances

Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

doi: 10.1126/sciadv.adl6082

Figure Lengend Snippet: ( A ) Control, p37 KO, and p37 KO HeLa cells reconstituted with p37-FLAG were treated with 400 nM BafA1 for 4 hours, followed by immunostaining for LC3 and FLAG; n = 3, one-way ANOVA ( P = 0.004) with post hoc Tukey test. ( B ) iNeurons treated with lentiviral-delivered p37 shRNA (#81 or #83) for 4 days were treated with 400 nM BafA1 for 6 hours; n = 4, one-sample t test. ( C ) Control, p37 KO, and p37 KO with p37-FLAG–expressing HeLa cells were incubated in EBSS for 2 hours, followed by immunostaining for PI(3)P; n = 3 to 4, one-sample t test and two-tailed unpaired Student’s t test. ( D and E ) Endogenous ATG14L was immunoprecipitated from control and p37 KO HeLa cells (D) or control and p37-Clover–overexpressing HeLa cells (E); n = 4 to 5; one-sample t test. ( F to H ) Control or p37-FLAG–overexpressing cells were incubated in EBSS for 2 hours (F and G) and treated with 5 μM CB-5083 for 3 hours (G) or 400 nM BafA1 for 4 hours (H) where indicated, followed by immunostaining for PI(3)P (F), WIPI2 (G), or LC3 (H); n = 3, two-tailed unpaired Student’s t test. ( I ) HeLa cells expressing WT or SHP mutant p37-FLAG for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours; n = 3 to 4, one-sample t test. ( J ) Control or Beclin-1 KO HeLa cells expressing p37-FLAG were treated with 400 nM BafA1 for 4 hours; n = 5, one-sample t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques: Control, Immunostaining, shRNA, Expressing, Incubation, Two Tailed Test, Immunoprecipitation, Mutagenesis, Transfection, Over Expression, Construct, Knockdown

Journal: Science Advances

Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

doi: 10.1126/sciadv.adl6082

Figure Lengend Snippet: ( A ) Mouse striatal cells with WT (Q7/Q7) or mutant (Q111/Q111) huntingtin overexpressing p37-FLAG were analyzed by western blotting; n = 3, one-sample t test. ( B ) HTTQ74-EGFP aggregates in control and p37-overexpressing cells in control or ATG16 KO HeLa cells; n = 3, two-tailed paired Student’s t test. ( C and D ) A53T-SNCA-EGFP HeLa cells overexpressing p37-FLAG, p47-FLAG, or VCP-HA (C) or overexpressing WT or SHP mutant p37 (D) were analyzed by FACS; n = 5, one-sample t test. ( E ) Control, p37 KO, and p37 KO expressing p37-FLAG HeLa cells were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 3, one-way ANOVA ( P = 0.0074) with post hoc Tukey test. ( F ) A53T-SNCA-EGFP HeLa cells were treated with control or p37 siRNA for 48 hours, followed by FACS analysis; n = 3, one-sample t test. ( G ) Control and ATG16 KO HeLa cells overexpressing p37-FLAG were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 4, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( H ) Control and p37-FLAG–overexpressing HeLa cells pre-treated with 5 μM CB-5083 or DMSO for 1 hour were treated with puromycin for 4 hours, followed by immunostaining for ubiquitin-positive structures; quantification of the total area of ubiquitin-positive foci; n = 3, two-tailed paired Student’s t test. ( I ) Quantification of total, cytosolic, and nuclear Ub + inclusions in control and p37-overexpressing cells; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. Scale bars, 10 μm. *, unspecific band; SE, short exposure. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques: Mutagenesis, Western Blot, Control, Two Tailed Test, Expressing, Immunostaining, Ubiquitin Proteomics, Transfection, Over Expression, Construct, Knockdown

Journal: Science Advances

Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

doi: 10.1126/sciadv.adl6082

Figure Lengend Snippet: ( A ) Control, p37 KO, p37 KO expressing WT p37-FLAG, and p37 KO expressing WT p47-FLAG HeLa cells were immunostained for VCP and FLAG; n = 3, one-way ANOVA ( P < 0.0001) with post hoc Tukey test. ( B ) Cytosolic and nuclear fractions from control, p37 KO, and p37 KO expressing WT or SHP mutant p37-FLAG HeLa cells were analyzed for VCP protein levels with Lamin B1 as a nuclear marker and GAPDH as a cytosolic marker; n = 4, one-sample t test and two-tailed paired Student’s t test (for nuclear p37 KO analysis). ( C and D ) iNeurons treated with lentiviral-delivered shRNA#81 against p37 (C) or expressing lentiviral-delivered p37-Clover (D) for 4 days were immunostained for VCP and p37; n = 3, two-tailed paired Student’s t test. ( E ) HeLa cells incubated in EBSS for 6 hours were immunostained for VCP and analyzed for VCP signal in cytosol and nucleus; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques: Control, Expressing, Mutagenesis, Marker, Two Tailed Test, shRNA, Incubation, Transfection, Over Expression, Construct, Knockdown

Journal: Science Advances

Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

doi: 10.1126/sciadv.adl6082

Figure Lengend Snippet: ( A ) HeLa cells expressing WT or SHP mutant p37-FLAG were immunostained for ubiquitin; n = 3, one-way ANOVA ( P = 0.0001) with post hoc Tukey test. ( B ) Control and p37-FLAG–overexpressing HeLa cells were treated with CHX, followed by isolation of the nuclear fraction, n = 3, two-tailed paired Student’s t test. ( C ) Nucleoplasm and chromatin fraction isolated from control and p37 KO HeLa cells. ( D ) Control and p37 KO HeLa cells were treated with CHX, followed by isolation of the nuclear fraction, n = 3, two-tailed paired Student’s t test. ( E and F ) Control, p37 KO, and p37 KO HeLa cells expressing either WT or SHP mutant p37-FLAG were treated with mitomycin C (1 μg/ml) for 2 hours, followed by immunostaining; statistical analysis in (F); n = 3, one-way ANOVA ( P = 0.0049) with post hoc Tukey test. ( G and H ) iNeurons treated with lentiviral-delivered shRNA#83 against p37 (G) or expressing lentiviral-delivered p37-Clover (H) were treated with mitomycin C (1 μg/ml) for 6 hours, followed by immunostaining; two-tailed paired Student’s t test; n = 5 for (G); n = 3 for (H). ( I and J ) Control and p37 KO HeLa cells treated with mitomycin C (1 μg/ml) for 2 hours in the presence or absence of 5 μM CB-5083 were immunostained for 53BP1; quantification of the total area of 53BP1 foci in (J); n = 3, two-tailed paired Student’s t test. ( K ) Control and p37 KO cells were treated with mitomycin C (1 or 2 μg/ml) for 32 hours, and cell death was monitored every 4 hours; data are represented as slope values of cell death curve over time; n = 4 to 5, one-way ANOVA ( P = 0.0091) with post hoc Tukey test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments we used nontargeting control siRNAs.

Techniques: Expressing, Mutagenesis, Ubiquitin Proteomics, Control, Isolation, Two Tailed Test, Immunostaining, shRNA, Transfection, Over Expression, Construct, Knockdown

Journal: Science Advances

Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

doi: 10.1126/sciadv.adl6082

Figure Lengend Snippet: ( A and B ) Control, heterozygous VCP R159H mutant (WT/R159H), homozygous VCP R159H mutant (R159H/R159H), and control revertant (Rev) iNeurons were immunostained for VCP (A) or cytosolic and nuclear fractions were isolated (B); quantification of nuclear VCP signal in (A), n = 3, two-tailed paired Student’s t test; n = 3 to 5 in (B), one-sample t test. ( C ) Control, heterozygous VCP R159H, and homozygous VCP R159H mutant iNeurons were treated with mitomycin C (1 μg/ml) for 60 hours to measure cell death; n = 4; one-sample t test. ( D ) Endogenous immunoprecipitation of VCP from control, heterozygous VCP R159H, homozygous VCP R159H mutant, and control revertant iNeurons. ( E and F ) Control, heterozygous VCP R159H, and homozygous VCP R159H mutant iNeurons were treated with lentiviral-delivered control or shRNA against p37 for 4 days and immunostained; quantification of nuclear VCP signal in (E), n = 3; quantification of γ-H2AX-Ser 319 intensity in (F), n = 4, two-tailed paired Student’s t test. ( G ) p37 coordinates the shuttling and local functions of VCP between the cytosol and nucleus. p47 binding to p37 prevents p37 proteasomal degradation. An increase in p37 levels leads to increased VCP localization in the cytosol, resulting in its enhanced function in ERAD and autophagy, but impaired function in CAD and DNA damage repair. Depletion of p37 promotes VCP nuclear localization and impairs VCP function in ERAD and autophagy but enhances its function in CAD and DNA damage repair. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

Techniques: Control, Mutagenesis, Isolation, Two Tailed Test, Immunoprecipitation, shRNA, Binding Assay, Transfection, Over Expression, Construct, Knockdown

Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspeciﬁc band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantiﬁcation of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for signiﬁcant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspeciﬁc band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantiﬁcation of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for signiﬁcant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Knock-Out, Immunoprecipitation, Control, Autoradiography, Pulse Chase, Knockdown, Isolation, Mass Spectrometry, Labeling, MTS Assay

Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

Techniques: Binding Assay, Mutagenesis, Generated, Incubation, Isolation, Western Blot, Residue, Variant Assay, Irradiation

Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Puriﬁed SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deﬁcient I3mut are shown. See also Figure S5.

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Puriﬁed SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deﬁcient I3mut are shown. See also Figure S5.

Techniques: Ubiquitin Proteomics, Incubation, Immunoprecipitation, Size-exclusion Chromatography, Migration, Binding Assay

Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos ﬂuorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos ﬂuorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

Techniques: Western Blot, Incubation, Binding Assay

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