13 s  (Addgene inc)

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: Figure 4 | p38c and p38d interact with mTOR through DEPTOR. (a) Endogenous mTOR, Raptor, Rictor, Sin-1 and DEPTOR co-immunoprecipitate with endogenous p38g. We immunoprecipitated p38g from WT and p38g/d / MEF lysates using specific antibodies; immunoprecipitates (IP), supernatants and total lysates were analysed by SDS–PAGE using the antibodies indicated. (b,c) p38g interacts with mTOR through DEPTOR. HEK-293 cells were transfected with HA-p38g, 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 p38g and p38d with DEPTOR in HEK-293 cells. HA-p38g or HA-p38d expression vectors were co-expressed with Flag-DEPTOR in HEK-293T cells. Anti-Flag immunoprecipitates were analysed by SDS–PAGE. (e) HA- p38d co-immunoprecipitates with endogenous p38g. p38g immunoprecipitates from HEK-293T cells transfected with HA-p38d 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 p38g and pcDNA3 HA human p38d, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38gD129A and pCMV Flag p38dF324S, 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: Figure 5 | p38c and p38d phosphorylate and downregulate DEPTOR protein levels. (a) Structural organization of DEPTOR, indicating p38g and p38d phosphorylation sites found by in vitro kinase assay. (b) p38g and p38d phosphorylate native DEPTOR on the canonical serine-proline MAPK phosphorylation residues in vivo. p38g or p38d 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 p38g and p38d mutants induce DEPTOR degradation. (c) Flag-DEPTOR was expressed in HEK-293T cells alone or together with constitutively active p38g and p38d mutants, singly or together. HEK-293T cells starved for 30 h were incubated for 9 h with 10 mM 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 p38g and p38d 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 p38g and p38d mutants. Fresh media without serum was added together with 10 mM 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 p38g and pcDNA3 HA human p38d, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38gD129A and pCMV Flag p38dF324S, kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Phospho-proteomics, 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: Figure 6 | p38c/d control cell size and protein synthesis through DEPTOR levels. (a) p38g/d / MEFs present altered serum-induced DEPTOR degradation. WT and p38g/d / 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) p38g/d / 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. ***Po0.001 (t-test). (c) p38g/d / MEFs have downregulated protein synthesis. SUnSET was performed by pulsing 10 min 10 mg 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. ***Po0.001 (t-test). (d) p38g/d-induced DEPTOR degradation by the proteasome. HELA cells co-transfected with active p38g and p38d mutants were serum-starved for 30 h. Cells were treated with MG132 (10 mM) or vehicle together with 10 mM cycloheximide (CHX) for 9 h, and were analysed by immunoblotting with the indicated antibodies. (e) Silencing DEPTOR in p38g/d / 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 mg 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 p38g/d / 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. **Po0.01; ***Po0.001 (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 p38g and pcDNA3 HA human p38d, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38gD129A and pCMV Flag p38dF324S, kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Control, Western Blot, 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: Figure 9 | Deptor cardiac specific expression reduces heart size. (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 ¼ 5–9). Data are means±s.e.m. (n ¼ 5). *Po0.05; **Po0.01 (t-test). (e–h) Cardiac-specific DEPTOR silencing in p38g/d / mice restores normal heart size. Hearts from WT, p38g/d / and p38g/d / 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 p38g/d / and from AAV-TnT-shDeptor injected p38g/d / mice. Bottom: representative staining with FITC-WGA (green). (h) Cardiomyocyte cross-sectional area quantified from WGA-stained hearts. (n ¼ 5–7). Data are means±s.e.m. (n ¼ 5). *Po0.05; **Po0.01; ***Po0.001 (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 p38g and pcDNA3 HA human p38d, kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, USA); and pCEFL Flag p38gD129A and pCMV Flag p38dF324S, kindly provided by David Engelberg (The Hebrew University of Jerusalem, Israel).

Techniques: Expressing, Injection, Western Blot, Staining