p66Shc mediates SUMO2-induced endothelial dysfunction
- Department of Internal Medicine, Division of Cardiovascular Medicine, University of Iowa, United States
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, United States
- Genome Editing Core Facility, University of Iowa, United States
- Regional Institute of Education-NCERT, India
- Department of Biochemistry and Molecular Biotechnology, Mass Spectrometry Facility, University of Massachusetts Chan Medical School, United States
Figures
Figure 1
p66Shc mediates small ubiquitin-like modifier 2 (SUMO2)-induced reactive oxygen species (ROS) production and inflammation in endothelial cells.
(A) Immunoblot for Shc and SUMO2 expression in HUVECs following p66Shc knockdown (si-p66Shc) and SUMO2 overexpression (Ad-HA-SUMO2). (B, C) Quantification of ROS (MitoSOX fluorescence) in HUVECs from A. *p<0.05; n=24–32; Student’s t-test. (D) Quantification of ROS in HUVECs following p66Shc knockdown (si-p66Shc) and SUMO2 overexpression (Ad-HA-SUMO2). ***p<0.001, p<0.05; n=10–15; One-way ANOVA. Data represents mean ± SEM. Immunoblots are representative of at least three independent experiments. GC, scrambled siRNA used as control; HUVECs, human umbilical vein endothelial cells.
-
Figure 1—source data 1
PDF file containing original western blots for Figure 1.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig1-data1-v1.zip
-
Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig1-data2-v1.zip
Figure 2 with 2 supplements
Small ubiquitin-like modifier 2 (SUMO2) directly modifies p66Shc.
(A) Immunoblot for sumoylated p66Shc in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66Shc (Flag-p66Shc). (B) Immunoblot for p66Shc sumoylation in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66Shc (Flag-p66Shc) with or without overexpression of Ubc9 (HA-Ubc9) and SENP1 (Flag-SENP1). (C) Immunoblot for sumoylation of p66Shc in human umbilical vein endothelial cells (HUVECs) overexpressing SUMO2 (Ad-HA-SUMO2) or p66Shc (Ad-Flag-p66Shc). (D) Immunoblot for p66Shc sumoylation in HUVEC cells overexpressing SUMO2 (Ad-HA-SUMO2) or p66Shc (Ad-Flag-p66Shc) in the presence or absence of anacardic acid. Quantification of SUMO2-p66Shc from immunoblot of D. One-way ANOVA, followed by Tukey’s post-hoc analysis, n=4 *p<0.05, **p<0.01. (F) Immunoblot for sumoylated recombinant p66Shc in the presence of absence of Ubc9. Immunoblots are representative of at least three independent experiments. Data represents mean ± SEM. Ad-Lac Z was used as an adenoviral control to compensate for the difference in MOI among the groups.
-
Figure 2—source data 1
PDF file containing original western blots for Figure 2.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-data1-v1.zip
-
Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-data2-v1.zip
Figure 2—figure supplement 1
Small ubiquitin-like modifier 2 (SUMO2) promotes p66Shc SUMO2ylation in a dose-dependent manner.
Immunoblot of human umbilical vein endothelial cells (HUVECs) transduced with wild-type p66Shc (Ad-Flag-p66ShcWT) and SUMO2 (Ad-HA-SUMO2) at the indicated concentrations. Immunoblots are representative of at least three independent experiments.
-
Figure 2—figure supplement 1—source data 1
PDF file containing original western blots for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-figsupp1-data1-v1.zip
-
Figure 2—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
Immunoblot showing the relative overexpression level of p66Shc in human umbilical vein endothelial cells (HUVECs) used for (A) experiment 2 C and (B) experiment 2D.
-
Figure 2—figure supplement 2—source data 1
PDF file containing original western blots for Figure 2—figure supplement 2A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-figsupp2-data1-v1.zip
-
Figure 2—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 2A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-figsupp2-data2-v1.zip
-
Figure 2—figure supplement 2—source data 3
PDF file containing original western blots for Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-figsupp2-data3-v1.zip
-
Figure 2—figure supplement 2—source data 4
Original files for western blot analysis displayed in Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig2-figsupp2-data4-v1.zip
Figure 3 with 1 supplement
Small ubiquitin-like modifier 2 (SUMO2) modifies p66Shc at lysine-81.
(A) Modular structure of p66Shc showing conserved lysine-81 in the CH2 domain. (B) Tandem mass spectrometry of in vitro SUMO2ylated recombinant p66Shc. (C) Immunoblot showing sumoylation of p66Shc in HEK-293 cells overexpressing p66ShcWT (Flag-p66ShcWT) or p66ShcK81R (Flag-p66ShcK81R) with and without SUMO2 (HA-SUMO2). (D) Immunoblot showing sumoylation of p66Shc in human umbilical vein endothelial cells (HUVECs) overexpressing p66ShcWT (Ad-Flag-p66ShcWT) or p66ShcK81R (Ad-p66ShcK81R) with and without SUMO2 (Ad-HA-SUMO2). (E) Immunoblot for sumoylation of recombinant p66ShcWT or p66ShcK81R. (F) Immunoprecipitation of SUMO2-p66Shc using SUMO2-p66ShcK81 antibody or the matching IgG control in HUVECs followed by immunoblotting with SUMO2-p66ShcK81 antibody showing endogenous SUMO2ylation of p66Shc (left-side panel, the high-intensity IgG signal was masked with a tape which appears as a white strip). Immunoblotting with Shc antibody in whole-cell lysate (right-side panel). Immunoblots are representative of at least three independent experiments. CH2, collagen homology 2; PTB, phosphotyrosine binding; CH1, collagen homology 1; SH2, Src homology 2.
-
Figure 3—source data 1
PDF file containing original western blots for Figure 3C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data1-v1.zip
-
Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data2-v1.zip
-
Figure 3—source data 3
PDF file containing original western blots for Figure 3D.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data3-v1.zip
-
Figure 3—source data 4
Original files for western blot analysis displayed in Figure 3D.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data4-v1.zip
-
Figure 3—source data 5
PDF file containing original western blots for Figure 3E.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data5-v1.zip
-
Figure 3—source data 6
Original files for western blot analysis displayed in Figure 3E.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data6-v1.zip
-
Figure 3—source data 7
PDF file containing original western blots for Figure 3F.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data7-v1.zip
-
Figure 3—source data 8
Original files for western blot analysis displayed in Figure 3F.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-data8-v1.zip
Figure 3—figure supplement 1
Sumoylation of recombinant p66ShcWT for mass spectrometry.
(A) Coomassie stain of SDS-PAGE of recombinant p66ShcWT in its original form (not processed for SUMO2ylation), and after being SUMO2ylated and non-SUMO2ylated. (B) Silver stain of non-SUMO2ylated and SUMO2ylated recombinant p66ShcWT used for mass spectrometry.
-
Figure 3—figure supplement 1—source data 1
PDF file containing original western blots for Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-figsupp1-data1-v1.zip
-
Figure 3—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig3-figsupp1-data2-v1.zip
Figure 4
Small ubiquitin-like modifier 2 (SUMO2) promotes p66ShcS36 phosphorylation by sumoylation of K81.
(A) Immunoblot for p66ShcS36 phosphorylation in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66ShcWT (Flag-p66ShcWT). (B) Quantification of p66ShcS36 phosphorylation from A. *p<0.05; n=6; one-way ANOVA. (C) Immunoblot for p66ShcS36 phosphorylation in human umbilical vein endothelial cells (HUVECs) overexpressing SUMO2 (Ad-HA-SUMO2) or p66ShcWT (Ad-Flag-p66ShcWT). (D) Quantification of p66ShcS36 phosphorylation from C. *p<0.05; n=8; one-way ANOVA. (E) Immunoblot for p66ShcS36 phosphorylation in HUVECs expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT (Ad-Flag-p66ShcWT) or p66ShcK81R (Ad-Flag-p66ShcK81R). (F) Quantification of p66ShcS36 phosphorylation from E. *p<0.05, **p<0.01; n=11; two-way ANOVA. Immunoblots were quantified with iBright image analysis software. Data represents mean ± SEM. Immunoblots are representative of at least three independent experiments.
-
Figure 4—source data 1
PDF file containing original western blots for Figure 4A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig4-data1-v1.zip
-
Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig4-data2-v1.zip
-
Figure 4—source data 3
PDF file containing original western blots for Figure 4C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig4-data3-v1.zip
-
Figure 4—source data 4
Original files for western blot analysis displayed in Figure 4C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig4-data4-v1.zip
-
Figure 4—source data 5
PDF file containing original western blots for Figure 4E.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig4-data5-v1.zip
-
Figure 4—source data 6
Original files for western blot analysis displayed in Figure 4E.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig4-data6-v1.zip
Figure 5
p66ShcK81 SUMO2ylation of K81 in p66Shc promotes its translocation to the mitochondria.
(A) Immunoblot for p66ShcWT in whole-cell lysates or the mitochondrial fraction of human umbilical vein endothelial cells (HUVECs) ectopically expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT (Ad-Flag-p66ShcWT). (B) Quantification of the relative abundance of p66Shc in the mitochondria from A. **p<0.01, ****p<0.0001; n=5; one-way ANOVA. (C) Immunoblot for p66Shc in whole-cell lysates or the mitochondrial fraction of HUVECs ectopically expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT Ad-Flag-p66ShcWT or p66ShcK81R (Ad-Flag-p66ShcK81R). (D) Quantification of relative abundance of p66Shc in the mitochondria from C. *p<0.05, **p<0.01; n=7; two-way ANOVA. Data represents mean ± SEM. Immunoblots are representative of at least three independent experiments.
-
Figure 5—source data 1
PDF file containing original western blots for Figure 5A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig5-data1-v1.zip
-
Figure 5—source data 2
PDF file containing original western blots for Figure 5A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig5-data2-v1.zip
-
Figure 5—source data 3
PDF file containing original western blots for Figure 5C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig5-data3-v1.zip
-
Figure 5—source data 4
Original files for western blot analysis displayed in Figure 5C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig5-data4-v1.zip
Figure 6 with 1 supplement
Shc1K81R are resistant to small ubiquitin-like modifier 2 (SUMO2)-induced endothelial dysfunction.
(A) DNA sequencing data from a Shc1K81R mouse showing a point mutation (A>G) resulting in an amino acid change (K→R), and insertion of silent mutation to create a digestion site for ApaI endonuclease for genotyping. (B) DNA gel electrophoresis of a PCR-amplified and ApaI-digested amplicon identifying the Shc1K81R allele. (C, D) Representative immunoblots showing SUMO2 expression in aortic rings from wild-type and Shc1K81R mice (1.67×108 pfu/ring). (E, F) Graphs showing % contraction following induction of endothelium-dependent relaxation in aortic rings from (E) wild-type mice, n=14–16 from 4 mice and (F) Shc1K81R mice (n=15–16 rings from 4 mice) expressing a control vector (Ad-Lac Z) or overexpressing SUMO2 (Ad-HA-SUMO2). (G, H) Graphs showing % contraction following induction of endothelium-independent relaxation in aortic rings from (G) wild-type (n=14–16 from 4 mice) and (H) Shc1K81R mice (n=14–15 from 4 mice) expressing a control vector (Ad-Lac Z) or overexpressing SUMO2 (Ad-HA-SUMO2). Curves were compared for nonlinear fit. ****p<0.0001. Ach, acetylcholine; SNP, sodium nitroprusside; PE, Phenylephrine; Homo, homozygous; Het, heterozygous; and NTC, No template control. Data represents mean ± SEM.
-
Figure 6—source data 1
PDF file containing original western blots for Figure 6B.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-data1-v1.zip
-
Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6B.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-data2-v1.zip
-
Figure 6—source data 3
PDF file containing original western blots for Figure 6C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-data3-v1.zip
-
Figure 6—source data 4
Original files for western blot analysis displayed in Figure 6C.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-data4-v1.zip
-
Figure 6—source data 5
PDF file containing original western blots for Figure 6D.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-data5-v1.zip
-
Figure 6—source data 6
Original files for western blot analysis displayed in Figure 6D.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-data6-v1.zip
Figure 6—figure supplement 1
Physiological characterization of Shc1K81R mice.
(A) Immunoblot for p66Shc isoforms in aortic lysates from wild-type and Shc1K81R mice. (B) Body weight of Shc1K81R and wild-type mice (n=7/group). (C) Heart weight of Shc1K81R and wild-type mice (n=6/group). (D) Litter-size of Shc1K81R (n=7) and wild-type mice (n=8). Graphs showing % contraction following induction of (E) endothelium-dependent and (F) endothelium-independent relaxation in aortic rings from wild-type mice, (n=15 rings from 4 mice) and Shc1K81R mice (n=15 rings from 6 mice). Curves were compared for nonlinear fit. ***p<0.001. ns = not significant. Ach, acetylcholine; SNP, sodium nitroprusside; PE, Phenylephrine. Data represents mean ± SEM.
-
Figure 6—figure supplement 1—source data 1
PDF file containing original western blots for Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-figsupp1-data1-v1.zip
-
Figure 6—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig6-figsupp1-data2-v1.zip
Figure 7
p66ShcK81R knock-in (KI) prevents hyperlipidemia-induced endothelial dysfunction and oxidative stress.
(A) Immunoblot showing oxidized-LDL (o-LDL)-induced changes in small ubiquitin-like modifier 2/3 (SUMO2/3)-conjugated proteins in human umbilical vein endothelial cells (HUVECs) with and without SUMO2. (B) Graph showing serum cholesterol-level in wild-type, Ldlr-/- mice on a high fat diet, and Shc1K81R mice on a high fat diet (n=4/group). One-way ANOVA, ****p<0.0001. Graphs showing % contraction following induction of endothelium-dependent relaxation (C) and endothelium-independent relaxation (D) in aortic rings from male wild-type mice, (n=14 rings from 5 mice), Ldlr-/- mice on a high fat diet (n=18 rings from 6 mice) and Ldlr-/-:: Shc1K81R mice on a high fat diet (n=12 rings from 6 mice). Two-way ANOVA. ***p<0.001. (E) Representative images showing the level of 8-OHdG in aortic section of Ldlr-/- mice on a high fat diet (n=6 mice) and Ldlr-/-:: Shc1K81R mice on a high fat diet (n=6 mice) and their (F) quantification. One-way ANOVA. *p<0.05. HFD, high fat diet; Ach, acetylcholine; SNP, sodium nitroprusside; PE, Phenylephrine; 8-OHdG, 8 hydroxy deoxyguanosine. Data represents mean ± SEM.
-
Figure 7—source data 1
Source data for immunoblots in Figure 7A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig7-data1-v1.zip
-
Figure 7—source data 2
Original datasets for Figure 7A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig7-data2-v1.zip
Figure 8 with 1 supplement
p66ShcK81 sumoylation affects multiple key regulatory pathways in endothelial cells.
(A, B) Principal component analysis (PCA) of proteins expressed in endothelial cells expressing SUMO2+p66ShcWT and SUMO2+p66ShcK81R. (C, D) Bar chart presentation of key regulatory pathways differentially affected by small ubiquitin-like modifier 2 (SUMO2)-p66ShcWT (C) and SUMO2-p66ShcK81R (D) in endothelial cells. The protein expression data (fold change) were obtained from human umbilical vein endothelial cells (HUVECs) expressing SUMO2 with p66ShcWT or p66ShcK81R compared to those expressing Ad-Lac Z. Pathway enrichment analysis (B) was performed using R-based bioinformatics tools, including PROGENy/decoupleR pathway activity inference and visualization packages. Differentially regulated pathways associated with senescence, mitochondrial dysfunction, apoptosis signaling, and angiogenesis are shown. Data were derived from a sample size of n=6.
Figure 8—figure supplement 1
P66Shck81-SUMO2ylation induced changes in signaling pathways in human umbilical vein endothelial cells (HUVECs).
(A) Representative immunoblots of endothelial cell lysate expressing Flag-p66ShcWT, Flag-p66ShcK81R, and HA-SUMO2 (n=3 shown, n=6 performed). (B) Bubble chart representation of all the regulatory pathways impacted by SUMO2-p66Shc in endothelial cells, rescued by p66ShcK81R. Pathway enrichment analysis was performed using R-based Reactome pathway analysis and visualization packages on differential protein expression data obtained from HUVECs expressing SUMO2-p66ShcWT or SUMO2-p66ShcK81R compared with Ad-LacZ controls (n=6).
-
Figure 8—figure supplement 1—source data 1
PDF file containing original western blots for Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig8-figsupp1-data1-v1.zip
-
Figure 8—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/106344/elife-106344-fig8-figsupp1-data2-v1.zip
Author response image 1
Amplex Red assay performed in HUVECs with and without knockdown of p66Shc expressing SUMO2 (Ad-SUMO2) or a control virus (Ad-LacZ).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Shc1K81R mice | This paper | Shc1K81R | Whole-body knock-in of Shc1K81R |
| Genetic reagent (M. musculus) | LDLr-/- mice | Jax | B6.129S7-Ldlrtm1Her/J | LDL receptor knockout mice |
| Cell line (Homo sapiens) | HEK-293T | ATCC | Cat# CRL11268 | Shared by Dr. Dale E. Abel |
| Cell line (H. sapiens) | HUVEC | promocell | Cat# C1220 | Ordered through Fisher Scientific |
| Cell line (H. sapiens) | HUVEC | Lonza | Catalog #: C2519A | HUVEC - Human Umbilical Vein Endothelial Cells, Pooled |
| Antibody | anti-SUMO2/3 (Rabbit polyclonal) | Sigma-Millipore | Cat# 633306 | WB (1:1000) |
| Antibody | anti-SUMO2/3 (Rabbit polyclonal) | CST | Cat# 4971 | WB (1:1000) |
| Antibody | Anti-GAPDH | CST | Cat# 2118 S | WB (1:1000) |
| Antibody | Anti-HA (Mouse monoclonal) | CST | Cat# 3724 s | WB (1:1000) |
| Antibody | Anti-His (Mouse monoclonal) | CST | Cat# 2366 S | WB (1:1000) |
| Antibody | Anti-Ubc9 (Rabbit Monoclonal Antibody) | CST | Cat# XP 4786 s | WB (1:1000) |
| Antibody | Anti-Shc | BD | Cat # 610879 | WB (1:2000) |
| Antibody | anti-human phopsho-p66ShcS36 (Mouse monoclonal) | Abcam | Cat# Ab267413 | WB (1:1000) (This product is discontinued from abcam) |
| Antibody | anti-human SUMO2-p66ShcK81 (Rabbit polyclonal) | This paper | YenZym, CA | Detects p66Shc-sumoylation on non K81 motif as well |
| Antibody | anti-Flag (Mouse-monoclonal) | Sigma-Millipore | Cat# F1804 | WB (1:1000) |
| Antibody | anti-COXIV (Rabbit monoclonal) | CST | Cat# 4850 S | WB (1:1000) |
| Recombinant DNA reagent | HA-SUMO2 (plasmid) | This paper | Kumar Lab | |
| Recombinant DNA reagent | Flag-p66Shc (plasmid) | This paper | Kumar Lab | |
| Recombinant DNA reagent | FNpCDNA3 | Dr. Robert Oshima | Addgene plasmid # 45346 | |
| Recombinant DNA reagent | Flag-p66ShcK81R | This paper | Kumar Lab | |
| Recombinant DNA reagent | Flag-SENP1 | Dr. Edward Yeh | Addgene plasmid # 17357 | |
| Recombinant DNA reagent | Ubc9 | Dr. Edward Yeh | Addgene plasmid # 20082 | |
| Recombinant DNA reagent | Ad-Flag-p66ShcWT | This paper | Kumar Lab | |
| Recombinant DNA reagent | Ad-Flag-p66ShcK81R | This paper | Kumar Lab | |
| Recombinant DNA reagent | Ad-HA-SUMO2 | This paper | Kumar Lab | |
| Recombinant protein | Recombinant His-p66ShcWT | This paper | Kumar Lab | |
| Recombinant protein | Recombinant His-p66ShcK81R | This paper | Kumar Lab | |
| Commercial assay or kit | SUMO conjugation kit | Enzo | BML-UW8955-0001 | |
| Commercial assay or kit | Total cholesterol assay kit | Cell Biolab | STA-390 | |
| Other | Special diet a high fat diet | Envigo | TD.88317 | High fat diet |
| Other | Genotyping p66ShcK81R KI mice genotyping primer Forwards | This paper | PCR primers | CTT CGG AAT GAG TCT CTG TCA TC |
| Other | Genotyping p66ShcK81R KI mice genotyping primer Forwards | This paper | PCR primers | ACC CGA ACC AAG TAG GAA ACG |
| Sequence-based reagent | Shc1 | Thermo Fisher | Catalog #: 4390815 siRNA ID: s73683 | Silencer Select Pre-Designed siRNA |
| Sequence-based reagent | SUMO2 | Thermo Fisher | Catalog #: 4392421 siRNA ID: s13180 | Silencer Select Pre-Designed siRNA |
| Sequence-based reagent | siRNA: nontargeting control | Thermo Fisher | 4390844 | Silencer Select |
| Other | MitoSOX | Thermo Fisher | M36008 | Mitochondrial Superoxide Indicator. Final working concentration: 2 µM |
| Other | H2DCFDA | Thermo Fisher | D399 | Indicator for reactive oxygen species Final working concentration: 25 µM |
Additional files
-
Supplementary file 1
Data details for graphs presented in the manuscript.
- https://cdn.elifesciences.org/articles/106344/elife-106344-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/106344/elife-106344-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
p66Shc mediates SUMO2-induced endothelial dysfunction
eLife 14:RP106344.
https://doi.org/10.7554/eLife.106344.3
