Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a ) Immunoblot analysis of p38γ and p38δ activation and expression in heart extracts prepared from wild-type (WT) mice at different ages. ( b , c ) WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice were killed at 4, 6, 9 and 11 weeks. ( b ) Heart-weight-to-tibia-length ratio. ( c ) Cardiomyocyte cross-sectional area quantified in wheatgerm agglutinin (WGA)-stained hearts. Data are means±s.e.m. ( n =5–13). ** P <0.01; *** P <0.001 (two-way analysis of variance (ANOVA) coupled to Bonferroni post tests). ( d ) Top: representative haematoxylin and eosin staining of transverse heart sections from 9-week-old WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice. Bottom: Representative staining with FITC-WGA (green) in hearts from 9-week-old WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice. ( e ) Echocardiography results for 9-week-old WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice. IVS;d (inter-ventricular septum in diastole); LVPW;d (left ventricle posterior wall in diastole); LV (left ventricle). Data are means±s.e.m. ( n =5–13). * P <0.05; ** P <0.01; *** P <0.001 (one-way ANOVA coupled to Bonferroni post tests).

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Western Blot, Activation Assay, Expressing, Staining

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice were treated for 28 days with angiotensin II (AngII) (1 μg kg per minute) or saline, delivered by subcutaneously implanted osmotic minipumps. ( a ) Immunoprecipitation analysis of the phosphorylation and protein levels of p38γ and δ isoforms in heart extracts prepared from WT mice treated with AngII or saline. ( b ) Echocardiography results from AngII-treated WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice shown as the change relative to saline-treated controls. ( c ) Heart-weight-to-tibia-length ratios for WT, p38γ −/− , p38δ −/− and p38γ/δ −/− after 28 days of Ang II treatment. ( d , e ) Top: representative FITC-WGA staining (green) in hearts from 9-week-old WT, p38γ −/− , p38δ −/− and p38γ/δ −/− mice after AngII treatment. ( e ) Cardiomyocyte cross-sectional area quantified in WGA-stained hearts. ( f ) Echocardiography evaluation of systolic cardiac function increment after AngII treatment. IVS;d (inter-ventricular septum in diastole); LV (left ventricle). Data are means±s.e.m. ( n =6–12). ** P <0.01; *** P <0.001 (one-way analysis of variance coupled to Bonferroni post-tests); # P <0.05 ( t -test between indicated groups).

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Immunoprecipitation, Staining

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a , b ) Immunoblot analysis of mTOR signalling pathway activity ( a ) and the activation status of translation factors ( b ) in heart lysates of 9-week-old WT and p38γ/δ −/− mice. Bar charts show quantification of total protein or vinculin-normalized band intensities ( n =4). Data are means±s.e.m. * P <0.05; *** P <0.001 ( t -test). ( c ) In vivo measurement of protein synthesis. Mice were injected intraperitoneally with 0.040 μmol g −1 puromycin dissolved in 100 μl PBS. Exactly 30 min after injection, tissues were extracted and frozen in liquid N 2 for immunoblot analysis with anti-puromycin antibody ( n =4).

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Western Blot, Activity Assay, Activation Assay, In Vivo, Injection

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a ) Endogenous mTOR, Raptor, Rictor, Sin-1 and DEPTOR co-immunoprecipitate with endogenous p38γ. We immunoprecipitated p38γ from WT and p38γ/δ −/− MEF lysates using specific antibodies; immunoprecipitates (IP), supernatants and total lysates were analysed by SDS–PAGE using the antibodies indicated. ( b , c ) p38γ interacts with mTOR through DEPTOR. HEK-293 cells were transfected with HA-p38γ, Flag-DEPTOR or Myc-mTOR or a combination of these and immunoprecipitated with the indicated antibodies targeting the c-myc epitope ( b ) or Flag ( c ). Immunoblots were probed with the indicated antibodies. ( d ) Co-immunoprecipitation of p38γ and p38δ with DEPTOR in HEK-293 cells. HA-p38γ or HA-p38δ expression vectors were co-expressed with Flag-DEPTOR in HEK-293T cells. Anti-Flag immunoprecipitates were analysed by SDS–PAGE. ( e ) HA- p38δ co-immunoprecipitates with endogenous p38γ. p38γ immunoprecipitates from HEK-293T cells transfected with HA-p38δ were analysed by SDS–PAGE. IP, immunoprecipitation; TL, total lysate.

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Immunoprecipitation, SDS Page, Transfection, Western Blot, Expressing

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a ) Structural organization of DEPTOR, indicating p38γ and p38δ phosphorylation sites found by in vitro kinase assay. ( b ) p38γ and p38δ phosphorylate native DEPTOR on the canonical serine-proline MAPK phosphorylation residues in vivo . p38γ or p38δ active mutants were co-expressed in HEK-293T cells with Flag-DEPTOR or Flag-13xS/T→A DEPTOR (a mutated form with alanine substitutions of the S/T target residues). Flag-DEPTOR proteins were immunoprecipitated from cell lysates. Immunoprecipitates were analysed by SDS–PAGE and blotted with anti-phospho-threonine-proline and anti-phospho-serine-proline antibody; TL, total lysate. ( c , d ) Constitutively active p38γ and p38δ mutants induce DEPTOR degradation. ( c ) Flag-DEPTOR was expressed in HEK-293T cells alone or together with constitutively active p38γ and p38δ mutants, singly or together. HEK-293T cells starved for 30 h were incubated for 9 h with 10 μM cycloheximide (CHX) with or without 10% FBS. Cell lysates were analysed by immunoblotting with the indicated antibodies. ( d ) Endogenous DEPTOR levels are reduced when the active p38γ and p38δ mutants are overexpressed in HELA cells. HELA cells were serum-starved for 30 h and treated as in c . ( e ) DEPTOR phosphorylation mutants were expressed in HEK-293T alone or together with constitutively active p38γ and p38δ mutants. Fresh media without serum was added together with 10 μM cycloheximide (CHX) and cells collected at the times indicated. DEPTOR degradation was analysed by immunoblot.

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: In Vitro, Kinase Assay, In Vivo, Immunoprecipitation, SDS Page, Incubation, Western Blot

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a ) p38γ/δ −/− MEFs present altered serum-induced DEPTOR degradation. WT and p38γ/δ −/− MEFs were serum-starved for 30 h, followed by serum addition. Cells were collected at successive time points for immunoblotting with the indicated antibodies. ( b ) p38γ/δ −/− MEFs are of below-normal size. Cell size was measured by flow cytometry (forward scatter). Right: representative histogram. Left: quantification graph of the forward scatter mean fluourescence intensity (FSC-A MFI) relative to WT. Data are means±s.e.m. *** P <0.001 ( t -test). ( c ) p38γ/δ −/− MEFs have downregulated protein synthesis. SUnSET was performed by pulsing 10 min 10 μg ml −1 puromycin and chasing for 1 h before FACS analysis with anti-puromycin 12D10 antibody and anti-mouse IgG conjugated with PE. Data are means±s.e.m. *** P <0.001 ( t -test). ( d ) p38γ/δ-induced DEPTOR degradation by the proteasome. HELA cells co-transfected with active p38γ and p38δ mutants were serum-starved for 30 h. Cells were treated with MG132 (10 μM) or vehicle together with 10 μM cycloheximide (CHX) for 9 h, and were analysed by immunoblotting with the indicated antibodies. ( e ) Silencing DEPTOR in p38γ/δ −/− MEF cells restores mTOR signalling. MEFs were singly or doubly infected with two different DEPTOR lentiviral shRNA constructs for 24 h. Uninfected cells were eliminated by selection with 3 μg ml −1 puromycin for 1 week. The resulting cell lines were then serum-starved for 24 h before collecting. Equal amounts of whole-cell lysates were immunoblotted with the indicated antibodies. ( f ) Silencing of DEPTOR in p38γ/δ −/− MEFs increases protein synthesis. MEFs were infected as in e . In the resulting cell lines, the protein concentration per cell was measured by SUnSET assay, performed as in b . Data are means±s.e.m. ** P <0.01; *** P <0.001 (one-way analysis of variance coupled to Bonferroni post tests).

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Western Blot, Flow Cytometry, Transfection, Infection, shRNA, Construct, Selection, Protein Concentration

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a ) Endogenous cardiac Sin-1, mTOR, GβL, Rictor, Raptor and DEPTOR co-immunoprecipitate with endogenous p38γ. p38γ immunoprecipitates (IP), total lysates and Co-IP supernatants from WT heart lysates were analysed by SDS–PAGE. EB: beads with IgG control. ( b ) Endogenous cardiac DEPTOR co-immunoprecipitates with p38δ. Immunoblot analysis of p38δ immunoprecipitates (IP) and total lysates (TL) from the hearts of 9-week-old p38γ/δ −/− mice infected with AAV-TnT-p38γ act and AAV-TnT-p38δ act . ( c ) p38γ/δ −/− hearts express above-normal levels of DEPTOR protein. Immunoblot analysis of heart lysates from WT and p38γ/δ −/− mice starved for 4 h and re-fed for 2h ( n =3–4). ( d ) Cardiac DEPTOR levels during postnatal development correlate negatively with p38γ and p38δ activation and mTOR pathway activation. Heart lysates from WT p1 and 2-week-old mice were analysed by immunoblot ( n =6). ( e ) Levels of DEPTOR phosphorylation and ubiquitination in vivo are reduced in p38γ/δ −/− hearts. Upper panel: poly-ubiquitinated proteins were IP from WT and p38γ/δ −/− heart lysates and immunoprecipitates were immunoblotted with anti-DEPTOR antibody. Lower panels: WT and p38γ/δ −/− mice were intravenously injected with AAV-TNT-Flag-DEPTOR and hearts harvested at the age of 2 weeks. Flag-DEPTOR was immunoprecipitated from heart lysates, and immunoprecipitates were analysed by immunoblotting with the indicated antibodies. ( n =5). ( f ) Angiotensin II (ANGII) treatment induces a reduction in DEPTOR levels in WT hearts. WT mice were treated with ANGII or saline for 21 days. Heart lysates were analysed by immunoblotting. ( n =3). ( g – i ) MCKdelta KO mice have small hearts. ( f ) Heart-weight-to-tibia-length ratios in WT and MCKdelta KO (p38δ MCK−KO ) mice killed at 9 weeks. ( g ) Top: representative haematoxylin and eosin staining of transverse heart sections from 9-week-old WT and MCKdelta KO mice. Bottom: representative staining with FITC-WGA (green) in hearts from 9-week-old WT and MCKdelta KO mice. ( h ) Cardiomyocyte cross-sectional area quantified from WGA-stained hearts. ( j ) MCKdelta KO hearts have higher protein levels of DEPTOR. MCK-Cre control mice and MCKdelta KO (p38δ MCK−KO ) mice were starved for 4 h before being killed and tissue was collected. Heart lysates were analysed by immunoblotting; the bar chart shows quantification of vinculin-normalized band intensities (ImageJ; n =4). Data are means±s.e.m. ( n =5). ** P <0.01; *** P <0.001 ( t -test).

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Co-Immunoprecipitation Assay, SDS Page, Western Blot, Infection, Activation Assay, In Vivo, Injection, Immunoprecipitation, Staining

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a – d ) Cardiac-specific expression of active p38γ and p38δ mutant forms in p38γ/δ −/− hearts restores normal heart size. p38γ/δ −/− mice were intravenously injected at 4 weeks of age with AAV-TnT-p38γ act and AAV-TnT-p38δ act , and hearts were harvested from 9-week-old WT, p38γ/δ −/− and p38γ/δ −/− AAV-TnT-p38γ/δ act mice. ( a ) Immunoblot analysis of heart lysates. ( b ) Top: representative haematoxylin and eosin (H&E) staining of transverse heart sections. Bottom: representative staining with FITC-WGA (green). ( c ) Cardiomyocyte cross-sectional area quantified from WGA-stained hearts. ( d ) Heart-weight-to-tibia-length ratio. ( e – g ) Rapamycin treatment preserves normal heart size in MCKdelta KO hearts. MCK-Cre (control) and MCKdelta KO (p38δ MCK−KO ) mice were intraperitoneally injected daily with rapamycin (2 mg kg −1 per day) from 4 to 9 weeks of age. ( e ) Heart-weight-to-tibia-length ratio. ( f ) Top: representative H&E stained transverse heart sections from 9-week-old MCK-Cre and MCKdelta KO mice after rapamycin treatment. Bottom: representative FITC-WGA staining (green) in hearts from 9-week-old MCK-Cre and MCKdelta KO mice after rapamycin treatment. ( g ) Echocardiography analysis of 9-week-old MCK-Cre and MCKdelta KO mice treated with rapamycin or vehicle. IVS;d (interventricular septum in diastole); LVPW;d (left ventricle posterior wall in diastole); LV Mass (left ventricle) and ejection fraction. Data are means±s.e.m. ( n =5). * P <0.05; ** P <0.01; *** P <0.001 (one-way analysis of variance coupled to Bonferroni post test). NS, not significant.

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Expressing, Mutagenesis, Injection, Western Blot, Staining

Journal: Nature Communications

Article Title: p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

doi: 10.1038/ncomms10477

Figure Lengend Snippet: ( a – d ) Cardiac-specific expression of Flag-DEPTOR in WT hearts reduces heart size. WT mice were intravenously injected at 4 weeks of age with AAV-TnT-Flag-DEPTOR and hearts harvested at 9 weeks of age. ( a ) Immunoblot analysis of heart lysates. ( b ) Heart-weight-to-tibia-length ratio ( c ) Top: representative haematoxylin and eosin (H&E) staining of transverse heart sections from untreated (WT) and AAV-TnT-Flag-DEPTOR-injected mice. Bottom: representative staining with FITC-WGA (green). ( d ) Cardiomyocyte cross-sectional area quantified from WGA-stained heart. ( n =5–9). Data are means±s.e.m. ( n =5). * P <0.05; ** P <0.01 ( t -test). ( e – h ) Cardiac-specific DEPTOR silencing in p38γ/δ −/− mice restores normal heart size. Hearts from WT, p38γ/δ −/− and p38γ/δ −/− mice intravenously injected at birth with AAV-TnT-shDeptor were harvested at 9 weeks of age. ( e ) Immunoblot analysis of heart lysates. ( f ) Heart-weight-to-tibia-length ratio ( g ) Top: representative H&E staining of transverse heart sections from untreated WT and p38γ/δ −/− and from AAV-TnT-shDeptor injected p38γ/δ −/− mice. Bottom: representative staining with FITC-WGA (green). ( h ) Cardiomyocyte cross-sectional area quantified from WGA-stained hearts. ( n =5–7). Data are means±s.e.m. ( n =5). * P <0.05; ** P <0.01; *** P <0.001 (one-way analysis of variance coupled to Bonferroni post test). NS, not significant.

Article Snippet: The plasmids used in the different experiments were pRK5 myc Rat mTOR (plasmid #1861, Addgene); pRK5 FLAG human DEPTOR (plasmid #21334, Addgene); pRK5 FLAG human DEPTOR (13xS/T→A; plasmid #21702, Addgene); pRK5 FLAG DEPTOR (PDZ domain; plasmid #21701, Addgene); pRK5 FLAG DEPTOR (DEP domains; plasmid #21700, Addgene); pcDNA3-myc3-CUL1 (plasmid #19896, Addgene); pcDNA3-myc3-bTrCP (plasmid #20718, Addgene); HA-Ubiquitin (plasmid #18712); pcDNA3 HA human p38γ and pcDNA3 HA human p38δ, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38γ D129A and pCMV Flag p38δ F324S , kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Expressing, Injection, Western Blot, Staining

Journal: American journal of medical genetics. Part A

Article Title: Loss-of-Function of Endothelin Receptor Type A Results in Oro-Oto-Cardiac Syndrome

doi: 10.1002/ajmg.a.61531

Figure Lengend Snippet: (a) Schematic of the BRET assay for subcellular localization, in which EDNRA-Renilla luciferase 8 (RLuc8) is combined with Venus (V)-kras (plasma membrane), Venus-PTP1B (endoplasmic reticulum) or Venus-giantin (Golgi apparatus) to measure BRET in different cellular compartments. (b) Average net BRET of unstimulated HEK293 cells transfected with wildtype EDNRA-RLuc8 or EDNRA p.Q381P-RLuc8 and V-kras, V-PTPB1 or V-giantin. Assays were performed at least three times. Error bars represent SEM.; two-tailed t-test; n.s., not significant. (c) Confocal images of HEK293T cells transfected with mCherry-MEM and pcDNA3.1, wild type EDNRA or EDNRA p.Q381P and incubated with Hilyte fluor-488-ET-1. mCherry-MEM and Hilyte fluor-488-ET-1 co-localized within cells expressing wild type EDNRA or EDNRA p.Q381P but not in empty vector (mock)-transfected cells. Images are representative of ligand binding after a 5 minute incubation. Green and red channels represent Hilyte fluor-488-ET-1 and mCherry-MEM, respectively. (d) HEK293T cells transfected with empty vector (mock), wild type EDNRA or EDNRA p.Q381P were incubated with Hilyte fluor-488-ET-1 and quantitatively analyzed with a fluorescence microplate reader. Specific fluorescence was calculated by subtracting background fluorescence values (from empty wells treated with Hilyte fluor-488-ET-1) from the raw fluorescence values. Assays were performed in triplicate at least three times. Error bars represent SEM; two-tailed t-test *p < 0.05, **p< 0.01; n.s., not significant.

Article Snippet: These plasmids are referred to as pCMV-EDNRA (or wild type EDNRA) and pCMV-EDNRA p.Q381P. pEDNRA-RLuc8 was constructed by subcloning a myc-EDNRA fragment derived from pmyc-EDNRA-GFP (pmyc-ETA-GFP), a kind gift from Jeffery Walker, University of Wisconsin) using HindIII and AgeI . pEDNRA-RLuc8 p.Q381P was derived from EDNRA-RLuc8 using the QuickChange Lightning Site-Directed Mutagenesis Kit as described above. pcDNA3.1-VN-Gγ2, pcDNA3.1-VC-Gβ1, pNES-Venus-mG, pVenus-kras, pVenus-PTP1b, pVenus-giantin have been previously described ( Hollins, Kuravi, Digby, & Lambert, 2009 Masuho, 2015 #2272; Wan et al., 2018 ). pcDNA3-Gα q -HA was a kind gift by P. Wedegaertner ( Wedegaertner, Chu, Wilson, Levis, & Bourne, 1993 ). pcDNA3.1-masGRK3ct-Nluc was a kind gift from Kirill Martemyanov ( Masuho, Ostrovskaya, et al., 2015 ). mCherry-MEM was purchased from Addgene (plasmid 55779, deposited by Catherine Berlot) ( Yost, Mervine, Sabo, Hynes, & Berlot, 2007 ).

Techniques: Bioluminescence Resonance Energy Transfer, Luciferase, Transfection, Two Tailed Test, Incubation, Expressing, Plasmid Preparation, Ligand Binding Assay, Fluorescence

Journal: Cell death & disease

Article Title: FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

doi: 10.1038/s41419-023-05629-y

Figure Lengend Snippet: Fig. 3 FKBP51 promotes the K63-ubiquitination of Akt in melanoma cells through its TPR domain. a IP assay of A375 melanoma cells transfected with HA-Akt, HA-K63-Ub and Flag-FKBP51. Immunoprecipitated protein was then assayed by IB. Anti-HA antibody revealed an increased K63-linked ubiquitination of Akt upon FKBP51 overexpression. IB of whole lysates is also shown; γ-tubulin was used as loading control. b A375 melanoma WT and KO for FKBP51 were transfected with HA-Akt and HA-K63-Ub. Then, Akt was immunoprecipitated and assayed with anti-HA. IB assay showed an impaired Akt K63-ubiquitination in KO cells compared to WT cells. IB of whole lysates is also shown; γ-tubulin was used as loading control. c IP assay of A375 melanoma cells transfected with combinations of the following expressing vectors: HA-Akt, HA-K63-Ub, Flag-FKBP51, Flag-FKBP51s and Flag-mutTPR. IB assay with anti-HA revealed the K63-Ub residues bind to Akt, with exception of cells overexpressing FKBP51s or the protein mutant at the TPR domain. IB of whole lysates is also shown. d A375 WT and KO for FKBP51 were transfected with HA-Akt, HA-K63-Ub and HA-K63R-Ub. FKBP51 was rescued in KO cells, and Akt was then immunoprecipitated and assayed by IB with anti-HA. IB assay showed that FKBP51 overexpression restored Akt K63-ubiquitination only in presence of WT K63-Ub and not with mutated K63R-Ub. IB of whole lysates is also shown, with Hsp90 used as loading control. In all experiments, Akt was immunoprecipitated with anti-Akt antibody and IgG served as control for a non-specific binding. e In vitro ubiquitination assay with HA-IP from A375 melanoma cells previously transfected with HA-AKT + HA-TRAF6 + Flag-FKBP51 (lane 1), HA-AKT + HA-TRAF6 (lane 3) or EV (lane 2). The ubiquitination test was performed in presence of Ubc13/Uev1a as ubiquitin-conjugating (E2) enzymes. These proteins were added to ubiquitination reactions consisting of E1, ATP and Ub, as described in the experimental section. Ubiquitinated proteins were detected by IB with anti-ubiquitin antibody.

Article Snippet: PcDNA3 HA-tagged TRAF6 was a gift of Prof. Shao-Cong Sun (MD Anderson Cancer Center, Houston, TX, USA), while PcDNA3 HA-tagged PHLPP1 full length was purchased from Addgene (#37100) [37].

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Over Expression, Control, Expressing, Mutagenesis, Binding Assay, In Vitro

Journal: Cell death & disease

Article Title: FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

doi: 10.1038/s41419-023-05629-y

Figure Lengend Snippet: Fig. 4 PHLPP improves K63-ubiquitination of Akt. a IB assay of A375 cells transfected with siTRAF6 RNA and NS RNA as negative control. Cells were collected after 24 h from transfection. IB shows that silencing of TRAF6 decreased pAkt levels. b IB assay of A375 cells transfected with siPHLPP RNA and NS RNA as negative control. Cells were collected after 24 and 48 h from transfection. IB shows that silencing of PHLPP decreased TRAF6 levels. c IB analysis of A375 cells transfected with HA-Akt, HA-K63-Ub, HA-PHLPP and siPHLPP RNAs and immunoprecipitated with Akt. IgG served as control for non-specific binding. IB showed that PHLPP increased Akt K63-Ub binding, whereas silencing of the phosphatases decreased it. IB of whole lysates is also shown. d IP assay of A375 cells transfected with Flag-FKBP51, HA-PHLPP, HA-K63-Ub and siPHLPP RNAs. Cells were immunoprecipitated with a Flag antibody and IgG served as control for non-specific binding. Immunoprecipitated protein was then assayed by IB with anti-K63-Ub antibody. PHLPP increased K63-Ub binding to FKBP51. IB of whole lysates is also shown. e IP assay of A375 melanoma cells treated with 0, 0.5 and 1 µM of the HSP90 inhibitor 17-AAG for 16 h. Endogenous FKBP51 was immunoprecipitated with anti-FKBP51 antibody, while IgG served as control for a not-specific binding. IB showed that inhibition of HSP90 did not affect the binding of FKBP51 to PHLPP. Immunoblot of whole lysates is also shown. f IP assay of A375 melanoma cells transfected with Flag-FKBP51, FKBP51-mutPPIase or Flag-FKBP51-mutTPR. Flag-FKBP51 was immunoprecipitated with anti-Flag antibody, while IgG served as control for non-specific binding. Immunoprecipitated proteins were then assayed by IB, and anti-PHLPP antibody revealed that mutated TPR did not affect the binding of FKBP51 to PHLPP. IB of whole lysates is also shown.

Article Snippet: PcDNA3 HA-tagged TRAF6 was a gift of Prof. Shao-Cong Sun (MD Anderson Cancer Center, Houston, TX, USA), while PcDNA3 HA-tagged PHLPP1 full length was purchased from Addgene (#37100) [37].

Techniques: Ubiquitin Proteomics, Transfection, Negative Control, Immunoprecipitation, Control, Binding Assay, Inhibition, Western Blot

Journal: Cell death & disease

Article Title: FKBP51 plays an essential role in Akt ubiquitination that requires Hsp90 and PHLPP.

doi: 10.1038/s41419-023-05629-y

Figure Lengend Snippet: Fig. 6 Proposed mechanism for the interaction of FKBP51 with Akt and PHLPP in melanoma cells. Left, FKBP51 binds to PHLPP thus stabilizing TRAF6 and allowing the formation of a K63 polyubiquitin chain and the full activation of Akt. Right, PHLPP is not kept into the complex by FKBP51s, which hampers Akt ubiquitination and phosphorylation. The same occurs when PHLPP is subtracted from the FKBP51 complex.

Article Snippet: PcDNA3 HA-tagged TRAF6 was a gift of Prof. Shao-Cong Sun (MD Anderson Cancer Center, Houston, TX, USA), while PcDNA3 HA-tagged PHLPP1 full length was purchased from Addgene (#37100) [37].

Techniques: Activation Assay, Ubiquitin Proteomics, Phospho-proteomics

Journal: Cancer Science

Article Title: MicroRNA‐493‐5p‐mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

doi: 10.1111/cas.14292

Figure Lengend Snippet: Measurement of microRNA (miR)‐493‐5p and MYCN expression levels in clinical samples from hepatocellular carcinoma (HCC) patients. A,B, Relative expression levels of (A) miR‐493‐5p and (B) MYCN in clinical samples. Patients showing moderate to advanced hepatic tumors (length > 2.5 cm) were selected for the study. The box plots illustrate differential gene expression in 13 primary HCC tumors (T) compared with the corresponding nontumor tissues (NT). Mann–Whitney U test was used to calculate P values. C, Scatter plots of Spearman’s correlation coefficient analysis between miR‐493‐5p and MYCN relative expression, measured by real‐time quantitative PCR in all clinical samples (T and NT, N = 26). Red and blue plots show T and NT tissues, respectively

Article Snippet: For MYCN rescue experiments, the cells were incubated with 1.5 µg MYCN expression vector (plasmid #74163; Addgene) following the experimental procedure described above.

Techniques: Expressing, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: MicroRNA‐493‐5p‐mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

doi: 10.1111/cas.14292

Figure Lengend Snippet: Effect of MYCN knockdown and rescue on hepatic cancer cell growth and invasion. A, Hep3B cell growth assessment after MYCN silencing. Two distinct siRNAs were used to target MYCN (siMYCN_A and siMYCN_B). Scrambled siRNA was used as a negative control (siCtrl). Number of cells was estimated at the indicated times using a cell viability assay. B, Invasive abilities of Hep3B cells after MYCN knockdown. Cells that migrated through the Matrigel‐coated membrane were counted after 72 h. Photographs are representative of cell invasion for each condition. C, Cell viability. D, Invasion assay after MYCN and microRNA (miR)‐493‐5p overexpression in Hep3B cells (rescue experiment). MYCN expression vector did not contain MYCN mRNA 3′‐UTR. Cell viability was measured after 4 d. Validation of MYCN expression vector compared with the mock is shown in Figure . Data depicted in the figure show the mean ± SD. Significant differences were evaluated with a t test (n = 3). * P < .05, ** P < .01, *** P < .001

Article Snippet: For MYCN rescue experiments, the cells were incubated with 1.5 µg MYCN expression vector (plasmid #74163; Addgene) following the experimental procedure described above.

Techniques: Negative Control, Viability Assay, Invasion Assay, Over Expression, Expressing, Plasmid Preparation