length human lrp6 coding sequence  (Addgene inc)

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis.

doi: 10.7150/thno.48787

Figure Lengend Snippet: Figure 1. Cardiomyocyte-specific overexpression of LRP6 prevents cardiac dysfunction induced by pressure overload. (A), Echocardiographic analysis of fraction shortening (FS) in mice at different time-points (3d, 1w, 2w, and 4w) after TAC. *p < 0.05 vs Sham group; n = 6-10 mice/each group. (B), Western blot analysis of p-LRP6 and LRP6 levels in left ventricular tissues from mice at different time-points (3d, 1w, 2w, and 4w) after TAC. *p < 0.05; **p < 0.01 vs sham group; n = 4 mice/ each group. (C), Summary of the generation of tamoxifen-inducible cardiac-specific LRP6 overexpressing mice. (D), Representative images of Western blot analysis of LRP6 expression in heart and kidney tissues from LRP6CAG/MCM after injection of tamoxifen or DMSO for 3 consecutive days. (E), Representative images of Western blot analysis of LRP6 expression in isolated CMs and non-CMs from tamoxifen-injected LRP6CAG/MCM (LRP6 Over) or –MCM (MCM) mice. CMs: cardiomyocytes. (F), Overall strategy of exploring the effects of cardiac LRP6 overexpression in TAC mice. (G), Echocardiographic analysis of cardiac function in tamoxifen-injected LRP6CAG/MCM (LRP6 Over) or –MCM (MCM) mice at 2 weeks and 4 weeks after TAC or sham operation. FS: fraction shortening; EF: ejection fraction; LVID;d: left ventricular diastolic internal diameter. HR: heart rate; *p < 0.05; **p < 0.01; n = 7-12/each group.

Article Snippet: The full-length human LRP6 coding sequence was isolated from LRP6-pCS2-VSVG (Addgene 27282#) and inserted into the downstream of this cassette to construct CAG-CAT-LRP6 which was then directly sequenced to verify successful insertion of the full-length human LRP6 coding sequence.

Techniques: Over Expression, Western Blot, Expressing, Injection, Isolation

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis.

doi: 10.7150/thno.48787

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific overexpression of LRP6 attenuates cardiac hypertrophy and fibrosis induced by pressure overload. (A), HE and Masson staining of cross heart sections. Scale bars, 2 mm. (B), Representative images of Masson staining heart tissue were showed and the cardiac interstitial and vascular fibrosis were quantitatively analyzed. Scale bars, 100 um. (C), Western blot analysis of p-LRP6, LRP6, Col1, Col3, α-SMA, MMP2, MMP9, TGF-β1, p-smad2/3 in heart tissues. *p < 0.05; **p < 0.01; ***p<0.001; n = 3-5/each group. (D), Quantitative analysis of the ratio of heart weight/body weight (HW/BW) and heart/tibia (HW/TL). *p < 0.05; **p < 0.01; ***p < 0.001; n = 4-7/each group. Tamoxifen-injected-LRP6CAG/MCM (LRP6 Over) or –MCM (MCM) mice were analyzed at 4 weeks after TAC or sham operation.

Article Snippet: The full-length human LRP6 coding sequence was isolated from LRP6-pCS2-VSVG (Addgene 27282#) and inserted into the downstream of this cassette to construct CAG-CAT-LRP6 which was then directly sequenced to verify successful insertion of the full-length human LRP6 coding sequence.

Techniques: Over Expression, Staining, Western Blot, Injection

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis.

doi: 10.7150/thno.48787

Figure Lengend Snippet: Figure 3. Cardiomyocyte-specific overexpression of LRP6 inhibits the expression of cardiomyocyte-derived-Wnt5a/11 under pressure overload. (A), Western blot analysis of LRP6 expression in CMs, and Col1, MMP2, MMP9, TGF-β1, α-SMA, and p-smad2/3 expression in CFs. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON), and the culture medium from these CMs was used to stimulate CFs. *p < 0.05; **p < 0.01; ***p < 0.001; n = 4/each group. (B), Western blot analysis of Wnt5a and Wnt11 expression in CMs and the culture medium. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON). **p < 0.01; ***p < 0.001; n = 5-6/each group. (C), Western blot analysis of Wnt5a and Wnt11 expression in tamoxifen-injected-LRP6CAG/MCM (LRP6 Over) or –MCM (MCM) mice after TAC or sham operation. **p < 0.01; n = 5/each group.

Article Snippet: The full-length human LRP6 coding sequence was isolated from LRP6-pCS2-VSVG (Addgene 27282#) and inserted into the downstream of this cassette to construct CAG-CAT-LRP6 which was then directly sequenced to verify successful insertion of the full-length human LRP6 coding sequence.

Techniques: Over Expression, Expressing, Derivative Assay, Western Blot, Control, Transfection, Injection

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis.

doi: 10.7150/thno.48787

Figure Lengend Snippet: Figure 6. Cardiomyocyte-specific overexpression of LRP6 promotes the degradation of Wnt5a and Wnt11 by interaction with LRP6 and CTSD during pressure overload. (A), Western blot analysis of LRP6 and CTSD expression in tamoxifen-injected LRP6CAG/MCM (LRP6 Over) or –MCM (MCM) mice at 4 weeks after TAC or sham operation. *p < 0.05; **p < 0.01; n = 4 mice/each group. (B), Western blot analysis of LRP6 and CTSD expression in control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6) with or without MS. ***p < 0.001; n = 4-6/each group. (C), Interaction of LRP6 and CTSD was analyzed by IP analysis in control (Ad-CON) or LRP6-overexpressing

Article Snippet: The full-length human LRP6 coding sequence was isolated from LRP6-pCS2-VSVG (Addgene 27282#) and inserted into the downstream of this cassette to construct CAG-CAT-LRP6 which was then directly sequenced to verify successful insertion of the full-length human LRP6 coding sequence.

Techniques: Over Expression, Western Blot, Expressing, Injection, Control

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis.

doi: 10.7150/thno.48787

Figure Lengend Snippet: Figure 9. Mutants of LRP6 or CTSD inhibit the interaction of LRP6 and CTSD. (A), Sequencing results of LRP6 and CTSD mutations. (B), Schematic diagram of LRP6 and CTSD showing approximate locations of the mutants. (C), IP analysis of the interaction among LRP6 WT, LRP6 (P1104S), or LRP6 (P1427Q) and CTSD WT or CTSD (G136R). LRP6 WT and CTSD WT or their mutant plasmids were transfected into 293T cells for 48 h. The experiment was repeated 4 times. (D), Western blot analysis of LRP6, CTSD, Wnt5a, Wnt11, and MESD in stretched COS7 cells transfected with LRP6 WT and CTSD WT or their mutant plasmids as in (C). ****P < 0.0001, n = 3/each group.

Article Snippet: The full-length human LRP6 coding sequence was isolated from LRP6-pCS2-VSVG (Addgene 27282#) and inserted into the downstream of this cassette to construct CAG-CAT-LRP6 which was then directly sequenced to verify successful insertion of the full-length human LRP6 coding sequence.

Techniques: Sequencing, Mutagenesis, Transfection, Western Blot

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis.

doi: 10.7150/thno.48787

Figure Lengend Snippet: Figure 10. Proposed model of cardiac protection mediated by cardiac-specific LRP6 overexpression in response to pressure overload. LRP6 overexpression suppresses β-catenin activation, which may contribute to the inhibition of cardiac hypertrophy. LRP6 overexpression inhibits cardiomyocyte-derived Wnt5a/Wnt11 to inhibit cardiac fibrosis. Cardiomyocyte-expressed LRP6 interacts with CTSD, which may promote the degradation of Wnt5a and Wnt11.

Article Snippet: The full-length human LRP6 coding sequence was isolated from LRP6-pCS2-VSVG (Addgene 27282#) and inserted into the downstream of this cassette to construct CAG-CAT-LRP6 which was then directly sequenced to verify successful insertion of the full-length human LRP6 coding sequence.

Techniques: Over Expression, Activation Assay, Inhibition, Derivative Assay

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 1. LRP6 is involved in myocardial ischemia reperfusion. Western blot analysis of LRP6, p-LRP6, p-AKT(T308), p-AKT (S473), p-GSK3α and p-GSK3β(S9) expres sion in ischemia area of heart tissue from mice subjected to I/R surgery at different reperfusion time points (15min, 30min, 1 h) after ischemia for 45min. A. Representative images. B. Quantitative analysis of p-LRP6 and LRP6 level. C. Quantitative analysis of p- AKT(T308) and p-AKT(S473) level. D. Quantitative analysis of p-GSK3α and p- GSK3β(S9) level. Values are the means ± S. E.M. N = 3/group. *p < 0.05, **p < 0.005.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Western Blot

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 2. Cardiac specific LRP6 overexpression protects heart from I/R injury. A. The overall experimental protocol of in vivo to explore the effects of cardiac LRP6 overexpression in mice subjected to I/R. 6–8 weeks age male LRP6-CTG or MCM mice were intraperitoneally injected with tamoxifen (30 mg/kg) for 3 continuous days respectively, 2 weeks later, these mice (LRP6-CTG mice and MCM mice) were subjected to I/R surgery or sham operation. The MCM mice were used as control group. B. The expression of LRP6 and p-LRP6 in MCM mice and LRP6-CTG mice were analyzed by western blot. N = 3/group. C. Echocardiographic analysis at 24 h after I/R surgery or sham operation in MCM and LRP6-CTG mice. Enjection fraction (EF) and fraction shortening (FS) were quantified at 24 h after I/R injury. N = 4–8/group. D. Radial strain analysis in MCM mice and LRP6-CTG mice at 24 h after I/R were obatined from VevoStrain analysis software. Upper lane: Representative images of radial strain curves. Colored lines represent 6 standard myocardial regions; 7th black line calculates average (global) strain at each time point. Down lane: Quantitative analysis of radial strain both globally and in infarct area (anterior apex), and maximal opposite wall delay in time. N = 6/group. E. Representative TTC staining of the heart sections from Evans blue perfused hearts was shown for each group. The bar = 500 μm. The ratio of infarct area (IA) to area at risk (AAR), and area at risk/left ventricle (AAR/LV) were quantified at 24 h after I/R injury, as detailed in the Methods. N = 12–15/group. Values are means ± S.E.M. *p < 0.05, **p < 0.005,***p < 0,001 and ****p < 0.0001 vs. MCM group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Over Expression, In Vivo, Injection, Control, Expressing, Western Blot, Software, Staining

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 3. Cardiac specific LRP6 overexpression inhibits cardiomyocytes apoptosis following I/R. A. Terminal deoxynucleotidyl transferase-mediated dUTP (deoxy uridine triphosphate)-biotin nick-end labeling (TUNEL) in heart tissue from MCM and LRP6-CTG mice at 24 h reperfusion following 45min ischemia. Scale bar, 50 μm. The percentage of TUNEL positive cells was calculated. N = 4/group. B–C. Western blot analysis of apoptosis-related proteins (cleaved-caspase3; p-JNK; JNK) expression and an ER stress marker protein (caspase12) in the ischemia zone of hearts from MCM and LRP6-CTG mice at 24 h reperfusion after 45min ischemia. N = 3/group. In vitro, neonatal rat cardiomyocytes were cultured and transfected Ad-LRP6 (LRP6 OVER) or control adenovirus (CON), 72 h later, 200 μM H2O2 was treated to induce oxidative stress, and PBS treatment was as control. D. TUNEL staining analysis of control or LRP6 overexpressed-cardiomyocytes treated with H2O2 for 24 h, scale bar, 200 μm. The percentage of TUNEL positive cells (apoptosis) was calculated. N = 11/group. E. Western blot analysis for apoptosis-related proteins (cleaved-caspase3; p-JNK; JNK) expression and ER stress marker protein (caspase12) expression in control or LRP6 overexpressed-cardiomyocytes treated with H2O2 or PBS for 24 h. N = 3/group. Values are means ± S.E.M. *p < 0.05, **p < 0.005; ***p < 0.001. and ****p < 0.0001.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Over Expression, End Labeling, TUNEL Assay, Western Blot, Expressing, Marker, In Vitro, Cell Culture, Transfection, Control, Staining

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 4. Cardiac specific LRP6overexpression promotes HSF1 nucleus translocation and phosphorylation of GSK3β and AMPK after I/R. A. Western blot analysis of LRP6, p-LRP6, p-AKT(T308), p-AKT(S473), AKT, p-GSK3β(S9), GSK3β, p-ERK, ERK in the ischemia zone of hearts from MCM and LRP6-CTG mice at 3 h reperfusion following 45min ischemia. N = 3/group. B–C. Western blot analysis of the active β-catenin and p-AMPK level in the ischemia zone of hearts from MCM and LRP6-CTG mice at 3 h reperfusion following 45min ischemia. N = 3/group. In vitro, human cardiomyocytes (AC16 cell line) were cultured and transfected LRP6 adenovirus (Ad- LRP6) or control adenovirus (Ad-CON), 72 h later, 200 μM H2O2 was treated to induce oxidative stress, and PBS treatment was as control. D. Western blot for p- AMPK, AMPK, p-GSK3β and GSK3β in control or LRP6 overexpressed-cardiomyocytes treated with 200 μM H2O2 or PBS for 3 h. N = 3/group. E. Western blot analysis of HSF1 in nucleus and cytoplasm lysates from the ischemia zone of hearts of MCM and LRP6-CTG mice at 3 h reperfusion following 45min ischemia. N = 3/group. F. Western blot analysis of HSF1 in nucleus and cytoplasm lysates from control or LRP6 overexpressed-cardiomyocytes treated with 200 μM H2O2 or PBS for 3 h. N = 3/ group. Values are means ± S.E.M. *p < 0.05, **p < 0.005,***p < 0.001 and ****p < 0.0001.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Translocation Assay, Phospho-proteomics, Western Blot, In Vitro, Cell Culture, Transfection, Control

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 5. Active GSK3β overexpression restrains the increased level of HSF1 and p-AMPK, and the inhibition of cardiomyocytes apoptosis mediated by LRP6 over expression in cardiomyocytes under oxidative stress. In vitro, neonatal rat cardiomyocytes were cultured and transfected LRP6 adenovirus (Ad-LRP6) and/or GSK3β- CA adenovirus (Ad-GSK3β-CA tagged with flag) or control adenovirus (Ad-CON), 72 h later, 200 μM H2O2 was treated to induce oxidative stress. A. Western blot analysis for the expression of flag, p-GSK3β(S9), GSK3β and LRP6 in cultured cardiomyocytes (grouped as above) treated with H2O2 for 24 h. N = 3/group. B. TUNEL staining of cultured cardiomyocytes (grouped as above) treated with H2O2 for 24 h. scale bar, 200 μm. The percentage of TUNEL positive cells was calculated. N = 10/group. C. Western blot analysis for p-JNK, JNK, cleaved-caspase3, and caspase12 in cultured LRP6 overexpressed-cardiomyocytes transfected with GSK3β-CA adenovirus (Ad-GSK3β-CA) or control adenovirus (Ad-CON), and these cardiomyocytes were treated with 200 μM H2O2 for 24 h. N = 3/group. D. Western blot for HSF1 in nucleus and cytoplasm, p-AMPK and AMPK in whole cell lysates from GSK3β-CA adenovirus (Ad-GSK3β-CA) and/or LRP6 adenovirus (Ad-LRP6) transfected cardiomycoytes treated with 200 μM H2O2 for 3 h. N = 3/group. Values are means ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Over Expression, Inhibition, In Vitro, Cell Culture, Transfection, Control, Western Blot, Expressing, TUNEL Assay, Staining

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 6. Si-HSF1 blunts the increase in p-GSK3β(S9) and p-AMPK level and attenuates the inhibition of apoptosis induced by LRP6 overexpression in cardiomyocytes during oxidative stress. A. Western blot analysis for HSF1 expression level in cultured cardiomyocytes with transfection of si-Scramble (si-Scram) or si-HSF1. N = 3/ group. B. The apoptosis was assessed by flow cytometry analysis for annexin-V and propidium iodide (PI) staining in cultured control or LRP6 overexpressed- cardiomyocytes treated with 200 μM H2O2 for 24 h, and these cardiomyocytes were pre-transfected into si-scramble (si-Scram) or si-HSF1. N = 3–6/group. C. Western blot analysis for p-AMPK, AMPK, p-GSK3β (S9) and GSK3β in cultured control or LRP6 overexpressed-cardiomyocytes treated with 200 μM H2O2 for 24 h, and these cardiomyocytes were pre-transfected into si-Scramble or si-HSF1. N = 3/group. A-C, Human cardiomyocytes (AC16 cells) were transfected control adenovirus (Ad-CON) or LRP6 adenovirus (Ad-LRP6) as control or LRP6 overexpressed-cardiomyocytes respectively. D. Western blot analysis for HSP70, p-GSK3β (S9) and GSK3β in cultured control or LRP6 overexpressed-cardiomyocytes (NRCMs) treated with 200 μM H2O2 for 3 h, and these cardiomyocytes were pre-treated with DMSO or KRIBB11(20 μM) for 48 h. N = 3/group. E. TUNEL staining of cultured cardiomyocytes (grouped as above) treated with H2O2 for 24 h. scale bar, 200 μm. The percentage of TUNEL positive cells was calculated. N = 4/group.Values are means ± S.E.M. *p < 0.05, **p < 0.005,***p < 0.001 and ****p < 0.0001.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Inhibition, Over Expression, Western Blot, Expressing, Cell Culture, Transfection, Flow Cytometry, Staining, Control, TUNEL Assay

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 7. AMPK inhibitor suppresses the increased level in p-GSK3β(S9) but not HSF1 induced by LRP6 over expression in cardiomyocytes during oxidative stress. Human cardiomyocytes (AC16 cells) were transfected control adenovirus (Ad-CON) or LRP6 adenovirus (Ad-LRP6) as control or LRP6 overexpressed- cardiomyocytes respectively. A. Western blot anal ysis for LRP6, p-AMPK, AMPK, p-GSK3β (S9), GSK3β in cultured control or LRP6 overexpressed- cardiomyocytes treated with 200 μM H2O2 for 3 h, and these cardiomyocytes were pre-treated with PBS or Compound C (20 μM) for 24 h. N = 3/Group. B. Western blot analysis of HSF1 in nucleus and cyto plasm in cultured control or LRP6 overexpressed- cardiomyocytes treated with 200 μM H2O2 for 3 h, and these cardiomyocytes were pre-treated with PBS or Compound C (20 μM) for 24 h. N = 3/group. Values are means ± S.E.M. *p < 0.05, **p < 0.005, ***p < 0.001.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Over Expression, Transfection, Control, Western Blot, Cell Culture

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 8. LRP6 overexpression inhibits the ubiquitina tion of HSF1 by regulation of GSK3β signaling in cardiomyocytes under oxidative stress. A. Real-time PCR analysis of HSF1 mRNA level in cultured car diomyocytes treated with 200 μM H2O2 for 3 h. N = 3/Group. Values are means ± S.E.M. ***p < 0.001. B. Analysis of the ubiquitination of HSF1 in car diomyocytes. Human cardiomyocytes (AC16 cells) were pre-transfected with control adenovirus (Ad- CON) or LRP6 adenovirus (Ad-LRP6), and then treated with 200 μM H2O2 or PBS for 3 h. C. The ubiquitination of HSF1 was analyzed in neonatal rat cardiomyocytes. These cardiomyocytes were pre- transfected with GSK3β-CA adnovirus (GSK3β-CA) and/or control adenovirus (Ad-CON) or LRP6 adenovirus (Ad-LRP6), and treated with 200 μM H2O2 for 3 h. D. The ubiquitination of HSF1 was analyzed in cardiomyocytes (AC16 cells). These car diomyocytes were pre-transfected with control adenovirus (Ad-CON) or LRP6 adenovirus (Ad-LRP6). After incubation of Compund C (20 μM) or PBS for 24 h, these cardiomyocytes were treated with 200 μM H2O2 for 3 h. B–D: Whole-cell lysates (WCL) were immunoprecipitated using an anti-HSF1 antibody, and the ubiquitin levels were determined by western blotting using an anti-ubiquitin antibody. Ubi, ubiq uitin. The experiments were repeated at least 3 times. E. The interaction between HSF1 and GSK3β was investigated by co-immunoprecipitation. HSF1 and GSK3β was precipitated from AC16 cell lysate with anti-HSF1 antibody and blotted with anti-GSK3β and p-GSK3β antibody, and vice versa. The experiments were repeated at least 3 times.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Cell Culture, Ubiquitin Proteomics, Transfection, Control, Incubation, Immunoprecipitation, Western Blot

Journal: Redox biology

Article Title: Low density lipoprotein receptor related protein 6 (LRP6) protects heart against oxidative stress by the crosstalk of HSF1 and GSK3β.

doi: 10.1016/j.redox.2020.101699

Figure Lengend Snippet: Fig. 9. The summary of the study. The present study indicated that cardiomyocyte-expressed-LRP6 inhibited ER stress and apoptosis by the crosstalk of HSF1 and GSK3β pathway which protects heart against myocardial I/R injury, and AMPK may be involved the process.

Article Snippet: The full-length human LRP6 coding sequence (from LRP6-pCS2-VSVG; Addgene 27282#) was inserted into the downstream of CAG-CAT to construct CAG-CAT-LRP6.

Techniques: