Anti-Glutathione Peroxidase 4/GPX4 Antibody Picoband®

Western blot analysis of Glutathione Peroxidase 4/GPX4 using anti-Glutathione Peroxidase 4/GPX4 antibody (A02059-1).
Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human HepG2 whole cell lysates,
Lane 2: human MCF-7 whole cell lysates,
Lane 3: human RT4 whole cell lysates,
Lane 4: monkey COS-7 whole cell lysates,
Lane 5: rat kidney tissue lysates,
Lane 6: rat testis tissue lysates,
Lane 7: mouse kidney tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Glutathione Peroxidase 4/GPX4 antigen affinity purified polyclonal antibody (Catalog # A02059-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for Glutathione Peroxidase 4/GPX4 at approximately 19 kDa. The expected band size for Glutathione Peroxidase 4/GPX4 is at 22 kDa.

Ferroptosis is present in CLP-induced AKI. Mice were subjected to CLP, then the kidneys were collected at the indicated time. A , B Shown are representative western blotting and quantification of GPX4 and PTGS2. C–E Quantitative analyses of MDA, GSH, and non-heme iron. F Quantitative analyses of Perl’s stain. G Representative Perl’s stain images. H Representative transmission electron microscopy (TEM) images. The black arrow indicates ferroptosis-like mitochondria. I Quantitative analyses of mitochondrial length. Mice were pretreated with liproxstatin-1 (10 mg/kg, ip) or DFO (20 mg/kg, ip) 0.5 h before being subjected to CLP. All kidney samples were collected at 24 h after CLP. J–K Shown are representative Hematoxylin-eosin (HE) stains and Periodic Acid-Schiff (PAS) stain. L Histological analyses of renal tubular injury. M Quantitative analyses of IHC stain of NGAL. N Representative immunohistochemistry (IHC) images for NGAL. O , P Quantitative analyses of Scr and BUN. n = 6 mice/group, mean ± SD were presented. *P < 0.05, **P < 0.01 and ns : P > 0.05 . ip: intraperitoneal
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Effects of MCTR1 on ferroptosis in CLP-induced AKI. Mice were given MCTR1 once, twice, or not during the 24 h experiment. The once-daily administration mode (qd): Mice were given MCTR1 (200 ng/mice, iv) 0.5 h before being subjected to CLP. The twice-daily administration mode (bid): Mice were given MCTR1 (200 ng/mice, iv) 0.5 h before being subjected to CLP, and then an additional MCTR1 (200 ng/mice, iv) was given 12 h after CLP. All kidney samples were collected at 24 h after CLP. A , B Shown are representative western blotting and quantification of GPX4 and PTGS2. C–E Quantitative analyses of MDA, GSH, and non-heme iron. F Quantitative analyses of Perl’s stain. G Representative Perl’s stain images. H Representative TEM images. The black arrow indicates ferroptosis-like mitochondria. I Quantitative analyses of mitochondrial length. J Histological analyses of renal tubular injury. K , L Shown are representative HE stains and PAS stain. M Representative IHC images for NGAL. N Quantitative analyses of IHC stain of NGAL. O , P Quantitative analyses of serum Scr and BUN. n = 6 mice/group, mean ± SD were presented. **P < 0.01 . iv: intravenous
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Effects of Nrf2 on MCTR1-regulated ferroptosis in CLP-induced AKI. Mice were given ML385 (30 mg/kg, ip, qd) for 7 d. On day 7, ML385 injection with or without a twice-daily administration mode of MCTR1 as described before in the following experiment. All kidney samples were collected at 24 h after CLP. A–D Shown are representative western blotting and quantification of Nrf2, GPX4, and PTGS2. E – G Quantitative analyses of MDA, GSH, and non-heme iron. H Quantitative analyses of Perl’s stain. I Representative Perl’s stain images. J Representative TEM images. The black arrow indicates ferroptosis-like mitochondria. K Quantitative analyses of mitochondrial length. L Histological analyses of renal tubular injury. M , N Representative HE stains and PAS stain. O Representative IHC images for NGAL. P Quantitative analyses of IHC stain of NGAL. Q , R Quantitative analyses of serum Scr and BUN. n = 6 mice/group, mean ± SD were presented. **P < 0.01
Index in PubMed under a CC BY license. PMID: 34961563

Effects of MCTR1 on LPS-induced lipid peroxidation and ferroptosis in vitro. A Viability curves for HK-2 cells treated with different concentrations (0, 1, 3, 10, 30, 100, and 300 nM) of MCTR1 and LPS (1 ug/ml) for 8 h. B Viability curves for HK-2 cells treated with different concentrations (0, 1, 3, 10, 30, 100, and 300 nM) of MCTR1 for 0 and 8 h. C HK-2 cells treated with MCTR1 (100 nM) and LPS (1 ug/ml) for 8 h, and the visualization of cell viability were evaluated by phase-contrast microscopy. D , E Representative western blotting and quantification of GPX4 and PTGS2 protein. F – H Quantitative analyses of MDA, GSH and, non-heme iron. I Quantitative analyses of oxidized C11-BODIPY 581/591 probe by flow cytometry. J Representative images of C11-BODIPY 581/591 fluorescent ratio-probe. K Representative TEM images. The black arrow indicates ferroptosis-like mitochondria. L Quantitative analyses of mitochondrial length. n = 3, mean ± SD were presented. **P < 0.01
Index in PubMed under a CC BY license. PMID: 34961563

Nrf2 is involved in the inhibitory effects of MCTR1 on LPS-induced ferroptosis. A , B HK-2 cells were treated with MCTR1 (100 nM) and LPS (1 ug/ml) for 8 h. Shown are representative western blotting and quantification of Nrf2. HK-2 cells were transfected with 30 nM Nrf2 siRNA for 48 h and then treated with MCTR1 (100 nM) and LPS (1 ug/ml) for 8 h. C , D Representative western blotting and quantification of Nrf2. E Visualization of cell viability were evaluated by phase-contrast microscopy. F Fold change of cell viability. G , H Representative western blotting and quantification of GPX4 and PTGS2. I – K Quantitative analyses of MDA, GSH, and non-heme iron. L Quantitative analyses of oxidized C11-BODIPY 581/591 probe by flow cytometry. M Representative images of C11-BODIPY 581/591 fluorescent ratio-probe. N Representative TEM images. The black arrow indicates ferroptosis-like mitochondria. O Quantitative analyses of mitochondrial length. n = 3, mean ± SD were presented. **P < 0.01
Index in PubMed under a CC BY license. PMID: 34961563

Western blot analysis of Glutathione Peroxidase 4/GPX4 using anti-Glutathione Peroxidase 4/GPX4 antibody (A02059-1).
Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human HCT116- WT whole cell lysates,
Lane 2: human HCT116-GPX4 KO whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. Then the membrane was incubated with rabbit anti-Glutathione Peroxidase 4/GPX4 antigen affinity purified polyclonal antibody (A02059-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for Glutathione Peroxidase 4/GPX4 at approximately 19 kDa. The expected band size for Glutathione Peroxidase 4/GPX4 is at 22 kDa.

IHC analysis of Glutathione Peroxidase 4/GPX4 using anti-Glutathione Peroxidase 4/GPX4 antibody (A02059-1).
Glutathione Peroxidase 4/GPX4 was detected in a paraffin-embedded section of human testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Glutathione Peroxidase 4/GPX4 Antibody (A02059-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of Glutathione Peroxidase 4/GPX4 using anti-Glutathione Peroxidase 4/GPX4 antibody (A02059-1).
Glutathione Peroxidase 4/GPX4 was detected in a paraffin-embedded section of mouse testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Glutathione Peroxidase 4/GPX4 Antibody (A02059-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Flow Cytometry analysis of THP-1 cells using anti-Glutathione Peroxidase 4/GPX4 antibody (A02059-1).
Overlay histogram showing THP-1 cells stained with A02059-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Glutathione Peroxidase 4/GPX4 Antibody (A02059-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.