jnk  (R&D Systems)

Journal: Cell Death and Differentiation

Article Title: Adaptor protein HIP-55-mediated signalosome protects against ferroptosis in myocardial infarction

doi: 10.1038/s41418-022-01110-z

Figure Lengend Snippet: A GST pull-down confirmed HIP-55 interaction with MAP4K1. HEK293A cells were co-transfected with GST or GST-HIP-55 with Flag-MAP4K1 plasmids. Cell lysates were immunoprecipitated with Glutathione-Sepharose 4B beads and then subjected to immunoblotting. n = 6. B Co-localization between HIP-55 and MAP4K1. NIH-3T3 cells were co-transfected with indicated plasmids for confocal photography. n = 6. C HIP-55 inhibited MAP4K1 kinase activity. Flag-tagged MAP4K1 was co-transfected with GST or GST-HIP-55 plasmids in HEK293A cells. MAP4K1 was purified with anti-FLAG beads to detect kinase activity by using an ADP-Glo™ Kinase Assay. n = 3. D Deficiency of HIP-55 significantly promoted MI-induced JNK over-activation. Representative immunoblots and statistical data of JNK activation in WT and HIP-55 −/− mice hearts after MI. n = 5. E Cardiac-specific overexpression of HIP-55 suppressed MI-induced JNK over-activation. Representative immunoblots and statistical data on JNK activation in WT and HIP-55 Tg mice hearts after MI. n = 5. F HIP-55 inhibited the JNK/GPX4 ferroptosis pathway. Cells were transfected with plasmids as indicated and then subjected to starvation for 24 h. Then the expression of GPX4 and phosphorylation of JNK were then detected. n = 6. * P < 0.05, ** P < 0.01. All error bars represent mean ± SEM.

Article Snippet: Antibodies to phospho-AKT (Ser473) (1:5000, #4060), pan-AKT (1:2000, #9272), GST (1:3000, #2624), HA (1:3000, #3724), phospho-JNK(T182/Y185) (1:2000, #4668), pan-JNK (1:2000, #9252), phospho-AKT substrate (1:5000, #9614) and GAPDH (1:5000, #5174) were from Cell Signaling Technology.

Techniques: Transfection, Immunoprecipitation, Western Blot, Activity Assay, Purification, Kinase Assay, Activation Assay, Over Expression, Expressing, Phospho-proteomics

Journal: Cell Death and Differentiation

Article Title: Adaptor protein HIP-55-mediated signalosome protects against ferroptosis in myocardial infarction

doi: 10.1038/s41418-022-01110-z

Figure Lengend Snippet: A Interaction assay between HIP-55 and seven isoforms of 14-3-3 proteins. HEK293A cells were co-transfected with the indicated 14-3-3 isoforms and GST-HIP-55. Interaction of HIP-55 with subtypes of 14-3-3 proteins was examined by GST pull-down. B Co-localization between HIP-55 and 14-3-3τ. NIH-3T3 cells were co-transfected with indicated plasmids for confocal photography. n = 6. C Phosphorylation of HIP-55 S269 and T291 were required for 14-3-3τ binding. HEK293A cells were co-transfected with the indicated vectors. GST pull-down was performed to detect interactions between 14-3-3τ with HIP-55WT or HIP-55AA. HIP-55AA mutation completely abolished the interaction between HIP-55 and 14-3-3τ. n = 3. D HIP-55 interacted with MAP4K1 in a S269/T291 phosphorylated-dependent manner. Cells were transfected with the indicated vectors and stimulated with EGF 100 ng/ml for 10 min. The interaction between HIP-55 and MAP4K1 was determined by Flag pull-down. n = 4. E Cells were transfected with the indicated vectors. Then the interaction between MAP4K1 and HIP-55WT or HIP-55AA was determined by GST pull-down. n = 4. F HIP-55 suppressed kinase activity of MAP4K1 in a S269/T291-dependent manner. Flag-MAP4K1 was co-transfected with GST-HIP-55WT or GST-HIP-55AA. Activity of MAP4K1 purified with anti-FLAG beads was detected by using ADP-Glo™ Kinase Assay. n = 3. G Cardiomyocytes were transfected as indicated and then treated with RSL3 (5 μM) for 24 h, and then imaged (magnification, ×10). n = 5. H Cell viability was determined by CCK-8 kit. n = 5. I Cardiomyocytes were transfected as indicated and then treated with RSL3 (5 μM) for 16 h, The expression of GPX4 was detected by immunoblot. n = 5. J HIP-55 inhibited JNK/GPX4 ferroptosis pathway in an S269/T291 phosphorylated-dependent manner. Cells were transfected with plasmids as indicated and then subjected to starvation for 24 h. Then the expression of GPX4 and phosphorylation of JNK were detected by immunoblot. n = 6. * P < 0.05, ** P < 0.01. All error bars represent mean ± SEM.

Article Snippet: Antibodies to phospho-AKT (Ser473) (1:5000, #4060), pan-AKT (1:2000, #9272), GST (1:3000, #2624), HA (1:3000, #3724), phospho-JNK(T182/Y185) (1:2000, #4668), pan-JNK (1:2000, #9252), phospho-AKT substrate (1:5000, #9614) and GAPDH (1:5000, #5174) were from Cell Signaling Technology.

Techniques: Transfection, Phospho-proteomics, Binding Assay, Mutagenesis, Activity Assay, Purification, Kinase Assay, CCK-8 Assay, Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Adaptor protein HIP-55-mediated signalosome protects against ferroptosis in myocardial infarction

doi: 10.1038/s41418-022-01110-z

Figure Lengend Snippet: A Genotyping of wildtype, HIP-55WT Tg and HIP-55AA Tg mice. B Western blots analysis of cardiac proteins from WT, HIP-55WT Tg , and HIP-55AA Tg mice displaying overexpression of HIP-55 in the transgenic hearts. C HIP-55 inhibited MI-induced JNK activation in a S269/T291-dependent manner. Western blots analysis of JNK phosphorylation in the heart tissues of WT, HIP-55WT Tg and HIP-55AA Tg mice after MI. n = 5. D HIP-55 inhibited MI-induced cardiomyocyte ferroptosis relies on its S269/T291 phosphorylation state. Representative immunoblots and statistical data of GPX4 expression in heart tissue from WT, HIP-55WT Tg , and HIP-55AA Tg mice after MI. n = 6. E Relative expression of MDA in heart tissue from WT, HIP-55WT Tg and HIP-55AA Tg mice after MI. n = 7. F Relative expression of GSH in heart tissue from WT, HIP-55WT Tg and HIP-55AA Tg mice after MI. n = 7. G Relative activity of SOD in heart tissue from WT, HIP-55WT Tg and HIP-55AA Tg mice after MI. n = 7. H HIP-55 suppressed myocardial infarction size depends on its S269/T291 phosphorylation. Representative Alcian blue-TTC staining and statistical data for area at risk and infarct size in heart tissue from WT, HIP-55WT Tg , and HIP-55AA Tg mice post-MI. WT, n = 9; HIP-55WT Tg , n = 11; HIP-55AA Tg , n = 9. I HIP-55 suppressed MI-induced cardiac contractile dysfunction in an S269/T291-dependent manner. Representative M-mode echocardiographic photographs (left) and cardiac contractile function (right) quantified by echocardiographic analysis of ejection fraction (EF) and fractional shortening (FS) in WT, HIP-55WT Tg , and HIP-55AA Tg mice after MI. n = 5–7. J HIP-55 alleviated cardiac hypertrophy after MI depends on its S269/T291 phosphorylation. The ratio of heart weight to tibial length (HW/TL) in WT, HIP-55WT Tg and HIP-55AA Tg mice. n = 6–7. * P < 0.05, ** P < 0.01. All error bars represent mean ± SEM. K The schematic diagram of HIP-55-dependent signalosome determines cardiomyocyte fate. HIP-55 functions as the central hub to assemble AKT, 14-3-3τ and MAP4K1 to form a signalosome, where AKT phosphorylates HIP-55 at its S269/T291 sites and HIP-55 then rewires AKT signaling to negatively regulate the MAP4K1 cell death pathway against ferroptosis and MI injury in a site-specific manner.

Article Snippet: Antibodies to phospho-AKT (Ser473) (1:5000, #4060), pan-AKT (1:2000, #9272), GST (1:3000, #2624), HA (1:3000, #3724), phospho-JNK(T182/Y185) (1:2000, #4668), pan-JNK (1:2000, #9252), phospho-AKT substrate (1:5000, #9614) and GAPDH (1:5000, #5174) were from Cell Signaling Technology.

Techniques: Western Blot, Over Expression, Transgenic Assay, Activation Assay, Phospho-proteomics, Expressing, Activity Assay, Staining