biotinylated ang ii  (Phoenix Pharmaceuticals)

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: Increased ferroptosis and ROS in the myocardium of Ang II-induced mice. A C57BL/6 mice were continuously subcutaneously infused with Ang II using ALZET® Osmotic Pumps (Model 1004) for 4 weeks. B Representative Western blot analysis of HO-1 and GPX4 levels in the myocardium of mice. GAPDH was used as a loading control. C Densitometric quantification of immunoblots in A (n = 6; *versus the Ctrl group; *P < 0.05, **P < 0.01). D Representative images of H&E staining, Sirius red staining and 4-HNE immunoreactivity in the myocardium of mice. E and F The levels of Fe 2+ in serum ( E ) and heart tissue. G-I Representative MDA content, SOD activity and GSH levels in the myocardium of mice. J C57BL/6 mice were fed a normal-iron diet (NID) or a high-iron diet (HID) and infused with Ang II for 4 weeks. K and M Body weight ( K ) and SBP ( M ) of Ang II + NID or Ang II + HID mice every 7 days (n = 6; *P < 0.05, **P < 0.01 versus the Ang II + NID group). N and O : ejection fraction (EF, N ) and fractional shortening (FS, O ), respectively. P Representative images of echocardiographic images and H&E and Sirius red staining and WGA staining in the myocardium of Ang II + NID or Ang II + HID mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Western Blot, Control, Staining, Activity Assay

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: Ferroptosis inhibitors protect against Ang II-induced cardiac dysfunction and remodeling in mice. A-D Echocardiographic analysis of the ejection fraction (EF, A ), fractional shortening (FS, B ), left ventricular posterior wall thickness in diastole (LVPWd, C ) and interventricular septal thickness at diastole (IVSd, D ). E Representative M-mode echocardiographic images of each group of mice. F The levels of Fe 2+ in serum. G, I and H Representative images of H&E and Sirius red staining and WGA in heart sections. J Quantification of the interstitial fibrotic area as determined by Sirius red staining. K Quantification of the size of myocardial cells as determined by WGA. L and N Representative Western blot analysis of β-MYHC, ANP, COL-1, MMP9 and TGF-β in cardiac tissues; GAPDH was used as a loading control. M and O Densitometric quantification of immunoblots in L and N, respectively (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II 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: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Staining, Western Blot, Control

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R expression was elevated in the myocardium of Ang II-infused mice and in cardiomyocytes. A Heatmap of P2X7R expression profiles identified in mouse models following Ang II infusion from the GSE89712 dataset (n = 2). B Representative immunoblot showing P2X7R in the myocardium of Ang II-infused mice. C mRNA levels of P2X7R in cardiac tissues. D Double immunofluorescence staining for P2X7R (red), the fibrosis marker vimentin (green, the top two rows) or the myocyte marker α-actin (green, the next two rows) in the myocardium of Ang II-infused mice. Merged images (orange) showing colocalization. E Quantification of the P2X7R immunoreactive area (%) in D . F Quantification of double immunoreactivity showing the percentages of P2X7R-positive plus α-actin- and vimentin-positive areas in images of Ang II-infused mice in D . G Representative immunoblot showing P2X7R in primary cardiomyocytes, H9c2 cardiomyocyte-like cell lines and primary fibroblasts stimulated with different concentrations of Ang II (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Vimentin 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: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Expressing, Western Blot, Double Immunofluorescence Staining, Marker

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R deficiency improved Ang II-induced cardiac diastolic and systolic dysfunction and cardiac remodeling in mice. A C57BL/6 or P2X7R knockout mice were infused with Ang II or saline for 4 weeks. Heart weight-to-body weight ratio (HW/BW) in each group of mice. B Representative M-mode echocardiographic images. C–F Echocardiographic analysis of the ejection fraction (EF, C ), fractional shortening (FS, D ), left ventricular posterior wall thickness in diastole (LVPWd, E ) and interventricular septal thickness at diastole (IVSd, F ). G The serum level of ANP was detected with an ELISA kit. H Representative H&E-stained images (longitudinal section, upper and transverse section, bottom). I and J Representative images of WGA in transverse sections of heart tissues ( I ) and quantification of the cardiomyocyte area from WGA ( J ). K and L Representative images of Masson staining ( K ) and quantification of the interstitial fibrotic area from Masson staining ( L ). M Representative Western blot analysis of β-MYHC, ANP, COL-1, MMP9 and TGF-β in cardiac tissues; GAPDH was used as a loading control. N Densitometric quantification of immunoblots in M (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Knock-Out, Saline, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Control

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R deficiency alleviates Ang II-induced myocardial ferroptosis in Ang II-induced mice. A Representative Western blot analysis of HO-1 and GPX4 levels in the heart tissues of mice. GAPDH was used as a loading control. B Densitometric quantification of immunoblots in A. C and D mRNA levels of HO-1 and GPX4 in heart tissues. E The contents of Fe 2+ in heart tissues measured by a kit. F Representative immunohistochemical images of 4-HNE in heart tissues. G Ultrastructural changes, including decreased mitochondrial volume, increased bilayer membrane density and the disappearance of mitochondrial cristae, were detected by transmission electron microscopy (TEM). H and I C57BL/6 or P2X7R knockout mice were fed a high-iron diet and infused with Ang II or saline for 4 weeks. Echocardiographic analysis of ejection fraction (EF, H ), fractional shortening (FS, I ) J-L Representative images of H&E ( J ) and Masson staining ( K ); quantification of interstitial fibrotic area from Masson staining ( L ) (n = 6; *P < 0.05, **P < 0.01 versus Ctrl group; #P < 0.05 versus Ang II group).

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Western Blot, Control, Immunohistochemical staining, Membrane, Transmission Assay, Electron Microscopy, Knock-Out, Saline, Staining

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R inhibition abolished Ang II-induced ferroptosis in vitro. H9c2 cells were pretreated with 10 μM A438079 (a P2X7R inhibitor) for 2 h and then stimulated with 1 μM Ang II for 24 h. A Representative Western blot analysis of HO-1 and GPX4 levels; GAPDH was used as a loading control. B and C Densitometric quantification of immunoblots in A . D-F Representative MDA content, SOD activity and GSH levels in each group. G The mitochondrial membrane potential of H9c2 cells was measured by JC-1 staining (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Inhibition, In Vitro, Western Blot, Control, Activity Assay, Membrane, Staining

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R blockade affects the stability of GPX4 and HO-1 mRNA by regulating HuR expression and nucleocytoplasmic shuttling. A and B H9c2 cells transfected with si-P2X7R were stimulated with 1 μM Ang Ⅱ for 24 h, and actinomycin D (Act D) was added at different times to interfere with the cell transcriptional cycle. The mRNA levels of GPX4 ( A ) and HO-1 ( B ) in H9c2 cells were detected. C Representative Western blot analysis of HuR levels with GAPDH as a loading control. D Densitometric quantification of immunoblots in C . E Immunofluorescence staining for HuR (green) in H9c2 cells. Increased fluorescence intensity in the cytoplasm after stimulation with 1 μM Ang Ⅱ. F and G, Relative GPX4 mRNA ( F ) and HO-1 mRNA ( G ) in H9c2 cells transfected with Si-HuR, stimulated with Ang Ⅱ and then treated with Act D. H and I RNA binding protein immunoprecipitation assays were used to verify the binding of HuR to GPX4 ( H ) and HO-1 ( I ) mRNA (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II 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: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Expressing, Transfection, Western Blot, Control, Immunofluorescence, Staining, Fluorescence, RNA Binding Assay, Immunoprecipitation, Binding Assay

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: Ang II directly binds to the P2X7R protein at positions LYS-66 and MET-212. A and B H9c2 cells were treated with 1 μM Ang II or biotinylated angiotensin II (Bio-Ang II) for 24 h. Representative Western blot analysis of P2X7R in cells with GAPDH as a loading control ( A ). Densitometric quantification of immunoblots in A ( B ) (n = 3; **P < 0.01 versus the Ctrl group). C H9c2 cells were treated with 1 μM Bio-Ang II or free biotin for 24 h, followed by double-immunofluorescence staining for biotin (green) and P2X7R (red). D and F Binding of Bio-Ang II to P2X7R was determined by pull-down assays. Bio-Ang II was added to streptavidin-agarose beads, and total lysates were used as an input control. Lysates prepared from heart tissues of mice infused with saline or Ang II were added to streptavidin-agarose beads with Bio-Ang II ( D ). Lysates were prepared from primary cardiomyocytes overexpressing Flag-P2X7R. Untreated beads (Blank), biotin alone (Bio) and unconjugated Ang II (Ang II) were used as controls ( E ). F Biolayer interferometry (BLI) analysis of the binding of Ang II to purified P2X7R protein. Ang II was added at different concentrations, and kinetic analysis was performed (shown in the panel below). G and H Molecular docking study between compound Ang II and the 3D structure of P2RX7 (PDB code: 6U9W ) ( G ). The key amino acids are connected to the molecular formula of Ang II through dashed lines ( H ). I HEK-293T cells were transfected with Flag-tagged P2X7R with THR-189 (Flag-PT189), Flag-tagged P2X7R with LYS-66 (Flag-PL66), Flag-tagged P2X7R with LYS-193 (Flag-PL193) and Flag-tagged P2RX7 with MET-212 (Flag-PM212). Lysates were added to streptavidin-agarose beads with Bio-Ang II. H Schematic representation of the key findings of this study. Ang II-induced myocardial remodeling involves upregulation of P2X7R expression and activation of ferroptosis. Intriguingly, Ang II directly interacts with the P2X7R protein. Furthermore, P2X7R mediates Ang II-induced myocardial ferroptosis through HuR, which affects the stability of GPX4 and HO-1 mRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Western Blot, Control, Double Immunofluorescence Staining, Binding Assay, Saline, Purification, Transfection, Expressing, Activation Assay

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: Increased ferroptosis and ROS in the myocardium of Ang II-induced mice. A C57BL/6 mice were continuously subcutaneously infused with Ang II using ALZET® Osmotic Pumps (Model 1004) for 4 weeks. B Representative Western blot analysis of HO-1 and GPX4 levels in the myocardium of mice. GAPDH was used as a loading control. C Densitometric quantification of immunoblots in A (n = 6; *versus the Ctrl group; *P < 0.05, **P < 0.01). D Representative images of H&E staining, Sirius red staining and 4-HNE immunoreactivity in the myocardium of mice. E and F The levels of Fe 2+ in serum ( E ) and heart tissue. G-I Representative MDA content, SOD activity and GSH levels in the myocardium of mice. J C57BL/6 mice were fed a normal-iron diet (NID) or a high-iron diet (HID) and infused with Ang II for 4 weeks. K and M Body weight ( K ) and SBP ( M ) of Ang II + NID or Ang II + HID mice every 7 days (n = 6; *P < 0.05, **P < 0.01 versus the Ang II + NID group). N and O : ejection fraction (EF, N ) and fractional shortening (FS, O ), respectively. P Representative images of echocardiographic images and H&E and Sirius red staining and WGA staining in the myocardium of Ang II + NID or Ang II + HID mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Western Blot, Control, Staining, Activity Assay

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: Ferroptosis inhibitors protect against Ang II-induced cardiac dysfunction and remodeling in mice. A-D Echocardiographic analysis of the ejection fraction (EF, A ), fractional shortening (FS, B ), left ventricular posterior wall thickness in diastole (LVPWd, C ) and interventricular septal thickness at diastole (IVSd, D ). E Representative M-mode echocardiographic images of each group of mice. F The levels of Fe 2+ in serum. G, I and H Representative images of H&E and Sirius red staining and WGA in heart sections. J Quantification of the interstitial fibrotic area as determined by Sirius red staining. K Quantification of the size of myocardial cells as determined by WGA. L and N Representative Western blot analysis of β-MYHC, ANP, COL-1, MMP9 and TGF-β in cardiac tissues; GAPDH was used as a loading control. M and O Densitometric quantification of immunoblots in L and N, respectively (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II 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: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Staining, Western Blot, Control

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R expression was elevated in the myocardium of Ang II-infused mice and in cardiomyocytes. A Heatmap of P2X7R expression profiles identified in mouse models following Ang II infusion from the GSE89712 dataset (n = 2). B Representative immunoblot showing P2X7R in the myocardium of Ang II-infused mice. C mRNA levels of P2X7R in cardiac tissues. D Double immunofluorescence staining for P2X7R (red), the fibrosis marker vimentin (green, the top two rows) or the myocyte marker α-actin (green, the next two rows) in the myocardium of Ang II-infused mice. Merged images (orange) showing colocalization. E Quantification of the P2X7R immunoreactive area (%) in D . F Quantification of double immunoreactivity showing the percentages of P2X7R-positive plus α-actin- and vimentin-positive areas in images of Ang II-infused mice in D . G Representative immunoblot showing P2X7R in primary cardiomyocytes, H9c2 cardiomyocyte-like cell lines and primary fibroblasts stimulated with different concentrations of Ang II (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Vimentin 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: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Expressing, Western Blot, Double Immunofluorescence Staining, Marker

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R deficiency improved Ang II-induced cardiac diastolic and systolic dysfunction and cardiac remodeling in mice. A C57BL/6 or P2X7R knockout mice were infused with Ang II or saline for 4 weeks. Heart weight-to-body weight ratio (HW/BW) in each group of mice. B Representative M-mode echocardiographic images. C–F Echocardiographic analysis of the ejection fraction (EF, C ), fractional shortening (FS, D ), left ventricular posterior wall thickness in diastole (LVPWd, E ) and interventricular septal thickness at diastole (IVSd, F ). G The serum level of ANP was detected with an ELISA kit. H Representative H&E-stained images (longitudinal section, upper and transverse section, bottom). I and J Representative images of WGA in transverse sections of heart tissues ( I ) and quantification of the cardiomyocyte area from WGA ( J ). K and L Representative images of Masson staining ( K ) and quantification of the interstitial fibrotic area from Masson staining ( L ). M Representative Western blot analysis of β-MYHC, ANP, COL-1, MMP9 and TGF-β in cardiac tissues; GAPDH was used as a loading control. N Densitometric quantification of immunoblots in M (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Knock-Out, Saline, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Control

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R deficiency alleviates Ang II-induced myocardial ferroptosis in Ang II-induced mice. A Representative Western blot analysis of HO-1 and GPX4 levels in the heart tissues of mice. GAPDH was used as a loading control. B Densitometric quantification of immunoblots in A. C and D mRNA levels of HO-1 and GPX4 in heart tissues. E The contents of Fe 2+ in heart tissues measured by a kit. F Representative immunohistochemical images of 4-HNE in heart tissues. G Ultrastructural changes, including decreased mitochondrial volume, increased bilayer membrane density and the disappearance of mitochondrial cristae, were detected by transmission electron microscopy (TEM). H and I C57BL/6 or P2X7R knockout mice were fed a high-iron diet and infused with Ang II or saline for 4 weeks. Echocardiographic analysis of ejection fraction (EF, H ), fractional shortening (FS, I ) J-L Representative images of H&E ( J ) and Masson staining ( K ); quantification of interstitial fibrotic area from Masson staining ( L ) (n = 6; *P < 0.05, **P < 0.01 versus Ctrl group; #P < 0.05 versus Ang II group).

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Western Blot, Control, Immunohistochemical staining, Membrane, Transmission Assay, Electron Microscopy, Knock-Out, Saline, Staining

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R inhibition abolished Ang II-induced ferroptosis in vitro. H9c2 cells were pretreated with 10 μM A438079 (a P2X7R inhibitor) for 2 h and then stimulated with 1 μM Ang II for 24 h. A Representative Western blot analysis of HO-1 and GPX4 levels; GAPDH was used as a loading control. B and C Densitometric quantification of immunoblots in A . D-F Representative MDA content, SOD activity and GSH levels in each group. G The mitochondrial membrane potential of H9c2 cells was measured by JC-1 staining (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Inhibition, In Vitro, Western Blot, Control, Activity Assay, Membrane, Staining

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: P2X7R blockade affects the stability of GPX4 and HO-1 mRNA by regulating HuR expression and nucleocytoplasmic shuttling. A and B H9c2 cells transfected with si-P2X7R were stimulated with 1 μM Ang Ⅱ for 24 h, and actinomycin D (Act D) was added at different times to interfere with the cell transcriptional cycle. The mRNA levels of GPX4 ( A ) and HO-1 ( B ) in H9c2 cells were detected. C Representative Western blot analysis of HuR levels with GAPDH as a loading control. D Densitometric quantification of immunoblots in C . E Immunofluorescence staining for HuR (green) in H9c2 cells. Increased fluorescence intensity in the cytoplasm after stimulation with 1 μM Ang Ⅱ. F and G, Relative GPX4 mRNA ( F ) and HO-1 mRNA ( G ) in H9c2 cells transfected with Si-HuR, stimulated with Ang Ⅱ and then treated with Act D. H and I RNA binding protein immunoprecipitation assays were used to verify the binding of HuR to GPX4 ( H ) and HO-1 ( I ) mRNA (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II 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: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Expressing, Transfection, Western Blot, Control, Immunofluorescence, Staining, Fluorescence, RNA Binding Assay, Immunoprecipitation, Binding Assay

Journal: Redox Biology

Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

doi: 10.1016/j.redox.2024.103154

Figure Lengend Snippet: Ang II directly binds to the P2X7R protein at positions LYS-66 and MET-212. A and B H9c2 cells were treated with 1 μM Ang II or biotinylated angiotensin II (Bio-Ang II) for 24 h. Representative Western blot analysis of P2X7R in cells with GAPDH as a loading control ( A ). Densitometric quantification of immunoblots in A ( B ) (n = 3; **P < 0.01 versus the Ctrl group). C H9c2 cells were treated with 1 μM Bio-Ang II or free biotin for 24 h, followed by double-immunofluorescence staining for biotin (green) and P2X7R (red). D and F Binding of Bio-Ang II to P2X7R was determined by pull-down assays. Bio-Ang II was added to streptavidin-agarose beads, and total lysates were used as an input control. Lysates prepared from heart tissues of mice infused with saline or Ang II were added to streptavidin-agarose beads with Bio-Ang II ( D ). Lysates were prepared from primary cardiomyocytes overexpressing Flag-P2X7R. Untreated beads (Blank), biotin alone (Bio) and unconjugated Ang II (Ang II) were used as controls ( E ). F Biolayer interferometry (BLI) analysis of the binding of Ang II to purified P2X7R protein. Ang II was added at different concentrations, and kinetic analysis was performed (shown in the panel below). G and H Molecular docking study between compound Ang II and the 3D structure of P2RX7 (PDB code: 6U9W ) ( G ). The key amino acids are connected to the molecular formula of Ang II through dashed lines ( H ). I HEK-293T cells were transfected with Flag-tagged P2X7R with THR-189 (Flag-PT189), Flag-tagged P2X7R with LYS-66 (Flag-PL66), Flag-tagged P2X7R with LYS-193 (Flag-PL193) and Flag-tagged P2RX7 with MET-212 (Flag-PM212). Lysates were added to streptavidin-agarose beads with Bio-Ang II. H Schematic representation of the key findings of this study. Ang II-induced myocardial remodeling involves upregulation of P2X7R expression and activation of ferroptosis. Intriguingly, Ang II directly interacts with the P2X7R protein. Furthermore, P2X7R mediates Ang II-induced myocardial ferroptosis through HuR, which affects the stability of GPX4 and HO-1 mRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: We used biotinylated Ang II (Bio-Ang II), which was created by Beijing Fanbo Biological Chemical Co., Ltd., and streptavidin magnetic beads (HY–K0208, MedChemExpress) for the pull-down assay.

Techniques: Western Blot, Control, Double Immunofluorescence Staining, Binding Assay, Saline, Purification, Transfection, Expressing, Activation Assay