Rabbit anti gpx4

Manufactured by Abcam

Sourced in United States

Rabbit anti-GPX4 is a primary antibody that specifically recognizes the glutathione peroxidase 4 (GPX4) protein. GPX4 is an antioxidant enzyme that plays a crucial role in protecting cells from oxidative damage.

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Anti-GPX4 Rabbit anti-

9 protocols using rabbit anti gpx4

Immunohistochemical Analysis of Formalin-Fixed Tissues

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Formalin-fixed, paraffin-embedded specimens were collected, and a routine H&E slide was first evaluated. Immunohistochemical staining was done on 5 μm-thick paraffin-embedded sections using mouse anti-Merlin (Abcam), rabbit anti-GPX4 (Abcam), rabbit anti-PTGS2 (Cell Signaling), mouse anti-Ki67 (Cell Signaling), rabbit anti-ACSL4 (Thermo Fisher), rabbit anti-TFRC (Abcam), and rabbit anti-YAP (Cell Signaling) antibodies with a standard avidin-biotin HRP detection system according to the instructions of the manufacturer (anti-mouse/rabbit HRP-DAB Cell & Tissue Staining Kit, R&D Systems Minneapolis, MN). Tissues were counterstained with haematoxylin, dehydrated, and mounted. In all cases, antigen retrieval was done with the BD Retrievagen Antigen Retrieval Systems as per instructions of the manufacturer.

Wu J., Minikes A.M., Gao M., Bian H., Li Y., Stockwell B.R., Chen Z.N, & Jiang X. (2019). Intercellular interaction dictates cancer cell ferroptosis via Merlin-YAP signalling. Nature, 572(7769), 402-406.

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Immunohistochemical Analysis of Formalin-Fixed Tissues

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Immunoblotting Analysis of Cellular Iron Homeostasis

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Unless otherwise stated, all chemicals including mouse monoclonal anti-β-actin were obtained from the Sigma Chemical Company, St. Louis, MO, USA. Mouse anti-human TfR1, Alexa Fluor 488 goat anti-rabbit IgG, TRIzol reagent, RPMI-1640 medium and fetal bovine serum were purchased from Invitrogen Life Technologies, Carlsbad, CA, USA; rabbit polyclonal anti-mouse Fpn1 from Novus Biologicals, Littleton, CO, USA; rabbit polyclonal anti-FTL (ferritin light chain) from Proteintech, Chicago, IL, USA; rabbit polyclonal anti-FTH (ferritin heavy chain) from Bioworld Technology Inc., Louis Park, MN, USA; rabbit anti-MAP2, rabbit anti-Aβ42, rabbit anti-HO1, rabbit anti-Gpx4, rabbit anti-ApoE, rabbit anti-IRP1 (iron regulatory protein 1) and rabbit anti-IRP2 (iron regulatory protein 2) from Abcam, Cambridge, MA, USA; and goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibody from LI-COR Biosciences, Lincoln, Nebraska, USA. AevertAid First Strand cDNA Synthesis Kit and BCA protein assay kits were bought from Thermo Scientific, Waltham, MA, USA; Faststart Universal SYBR Green Master and LightCycler96 from Roche, Nutley, NJ, USA; and protein RIPA lysis buffer from the Beyotime Institute of Biotechnology, Haimen, JS, China. All solutions were prepared fresh, prior to each assay.

Ma J., Guo Q., Shen M.Q., Li W., Zhong Q.X, & Qian Z.M. (2023). Apolipoprotein E is required for brain iron homeostasis in mice. Redox Biology, 64, 102779.

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Protein Expression Analysis of Kidney Injury Markers

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Total protein extracts from HK2 cells and mouse renal cortex tissues were prepared as previously described. Equal amounts of proteins were separated by SDS–PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). After being blocked with 5% skimmed milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with the following primary antibodies: mouse anti-KLF15 (Santa Cruz, CA, USA), rat anti-KIM-1, rabbit anti-SLC7A11 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-GPX4 (Abcam, Waltham, MA, USA), rabbit anti-NRF2 (Abcam), rabbit anti-KEAP1 (Proteintech, Rosemont, IL, USA), rabbit anti-Histone3 (H3, Proteintech), and rabbit anti-GAPDH (Proteintech). After being washed, the membranes were incubated with the secondary antibody for one hour. Finally, ECL plus chemiluminescence substrate was used to visualize the protein bands. ImageJ software v1.8.0 was used for relative protein quantification.

Yang X., Dong S., Fan Y., Xia Y., Yang F., Chen Z., Chen D., Zhang M., Liang D, & Zeng C. (2023). Krüppel-like Factor 15 Suppresses Ferroptosis by Activating an NRF2/GPX4 Signal to Protect against Folic Acid-Induced Acute Kidney Injury. International Journal of Molecular Sciences, 24(19), 14530.

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Immunohistochemical Analysis of Stem Cell Markers

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For histochemistry or immunohistochemistry, the tissues were fixed in 4% paraformaldehyde (PFA), dehydrated in a series of graded ethanol solutions, embedded in paraffin, and cut into 5 μm sections using a rotary microtome (Leica, Germany). The sections were then used for subsequent H&E staining or immunofluorescence microscopy. The antibodies used for immunohistochemistry were rabbit anti-CD133 (1:100 dilution, Proteintech), rabbit anti-ALDH1A1(1:100 dilution, Proteintech), rabbit anti-ABCG2 (1:100 dilution, Cell Signaling Technology), rabbit anti-β-catenin (1:100 dilution, Proteintech), rabbit anti-SOD2 (1:100 dilution, Proteintech) and rabbit anti-GPX4 (1:100 dilution, Abcam). Images were analyzed using the ImageJ program.

Zhu J., Li X., Liang C., Zhou X., Ge M., Chen Y., Jin J., Yin J., Xu H., Xie C, & Zhong C. (2021). Apatinib suppresses lung cancer stem-like cells by complex interplay between β-catenin signaling and mitochondrial ROS accumulation. Cell Death Discovery, 7, 102.

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Evaluation of GPX4 and H2A.X in PANC-1/GEM Cells

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PANC‐1/GEM cells were grown in 12‐well plates to achieve ≈80% confluence. Then, the participants were divided into groups and administered either a placebo or medication; the US group received US therapy for 2 min using a 1 W cm⁻² machine. The cells were permeabilized with 0.2% Triton X‐100 on ice for 20 min after being fixed with 4% paraformaldehyde overnight. This was followed by three 10‐min washes in 0.01 m phosphate‐buffered saline. Rabbit anti‐GPX4 (1:200; Abcam) and rabbit anti–H2A.X (1:100; Abcam) were incubated with the cells overnight at 4 °C after being blocked with 2% bovine serum albumin at room temperature for 1 h and rinsed with PBS three times for 10 min each. The cells were treated overnight at room temperature with Alexa Fluor 488 donkey anti‐rabbit antibody (1:1000; Abcam) and washed three times with 0.01 m PBS the following day. The cells were fixed in a medium containing 4′,6‐diamino‐2‐phenylindole (DAPI), and CLSM was used to reconstruct them in three dimensions after three washes in PBS.

Zhao Z., Wu Y., Liang X., Liu J., Luo Y., Zhang Y., Li T., Liu C., Luo X., Chen J., Wang Y., Wang S., Wu T., Zhang S., Yang D., Li W., Yan J., Ke Z, & Luo F. (2023). Sonodynamic Therapy of NRP2 Monoclonal Antibody‐Guided MOFs@COF Targeted Disruption of Mitochondrial and Endoplasmic Reticulum Homeostasis to Induce Autophagy‐Dependent Ferroptosis. Advanced Science, 10(30), 2303872.

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Western Blot Analysis of NSR Proteins

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NSR protein lysates were extracted 24 h post-intravitreal injection using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitors (Roche, Mannheim, Germany). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol. Lysates were studied by Western analysis as described previously [30 (link)]. Imaging was performed using GE Amersham Imager 600 (GE Healthcare, Chalfont St. Giles, UK). FIJI software was used for band densitometry [31 (link)]. Primary antibodies used were as follows: mouse anti-transferrin receptor (TfR, Invitrogen, Carlsbad, CA, USA), rabbit anti-Gpx4 (Abcam) and mouse anti-α-tubulin (Sigma-Aldrich). Secondary antibodies used were as follows: donkey anti-rabbit (ECL Rabbit IgG, HRP-linked whole antibody) and donkey anti-mouse (ECL Mouse IgG, HRP-linked whole antibody) (GE Healthcare, Chicago, IL, USA). All primary antibodies were used at a 1:1000 dilution, and all secondary antibodies were used at a 1:5000 dilution. α-tubulin served as an internal control.

Shu W., Baumann B.H., Song Y., Liu Y., Wu X, & Dunaief J.L. (2020). Ferrous but not ferric iron sulfate kills photoreceptors and induces photoreceptor-dependent RPE autofluorescence. Redox Biology, 34, 101469.

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Immunoblotting Analysis of Tight Junction and Oxidative Stress Proteins

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Equal amounts of protein were taken for per lane (20 µg) and separated by electrophoresis on 8-12% SDS-PAGE gels. Proteins were transferred to PVDF membranes by electrophoresis and sealed with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1.5 h at room temperature.
Thereafter, membranes were incubated with different primary antibodies at 4℃ overnight, including rabbit anti-Occludin-1 (1:1000, Invitrogen), mouse anti-Claudin-5 (1:1000, Invitrogen), and rat anti-ZO-1 (1:500, Santa Cruz Biotechnology), rabbit anti-MMP-9 (1:1000, Abcam), rabbit anti-SLC7A11 (1:1000, Abclone), rabbit anti-HO-1 (1:1000, Abclone), rabbit anti-GPX4 (1:1000, Abcam), rabbit anti-P2X7R (1:1000, Proteintech), rabbit anti-ERK1/2 (1:1000, Cell signaling technology), mouse anti-p-ERK1/2
(1:1000, Cell signaling technology), Rabbit anti-Albumin (1:1000, Proteintech), mouse anti-P53 (1:1000, Proteintech), rabbit anti-Transferrin (1:1000, Proteintech)and rabbit anti-GAPDH (1:3000, Abcam),. After washing with TBST for 3 times, PVDF membranes were immersed with horseradish peroxidase (HRP)conjugated secondary antibody (1:5000) for 1.5 h at room temperature. Immunolabeling were developed with enhanced ECL kit (Biosharp). Chemiluminescence levels was captured using an imaging system and data were normalized using GAPDH.

Liu C., Tian Q., Wang J., He P., Han S., Guo Y., Yang C., Wang G., Wei H, & Li M. (2023). Blocking P2RX7 Attenuates Ferroptosis in Endothelium and Reduces HG-induced Hemorrhagic Transformation After MCAO by Inhibiting ERK1/2 and P53 Signaling Pathways. Molecular neurobiology, 60(2).

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Ferroptosis Pathway Regulation Analysis

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The antibodies used were mouse anti-Ferritin Heavy Chain (Santa Cruz, sc-376594), rabbit anti-Ferritin Heavy Chain (Cell Signaling Technology, 4393), mouse anti-NCOA4 (Santa Cruz, 373739), rabbit anti-NCOA4 (Affinity, DF4255), rabbit anti-ATG5 (Cell Signaling Technology, 12994), rabbit anti-GPx4 (Abcam, 125066), rabbit anti-GPx4 (Proteintech, 14432) mouse anti-4-HNE (Abcam, ab48506), rabbit anti-HO-1 (Affinity, AF5393), rabbit anti-Nrf2 (Proteintech, 16396), rabbit anti-Transferrin receptor (Abcam, ab214039),and mouse anti-βactin (Proteintech, 66009). Antibody dilutions were according to manufacturer's instructions.
The following reagents were used: Deferoxamine (Sigma-Aldrich, D9533), Ferrostatin-1 (Medchemexpress, HY-100579), Necrostatin-1 (Medchemexpress, HY-15760), Z-VAD-FMK (Medchemexpress, HY-16658B), CQ (Medchemexpress, HY-17589A).

Zeng Z., Huang H., Zhang J., Liu Y., Zhong W., Chen W., Lu Y., Qiao Y., Zhao H., Meng X., Zou F., Cai S, & Dong H. (2022). HDM induce airway epithelial cell ferroptosis and promote inflammation by activating ferritinophagy in asthma. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(6).

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