The protein extracts prepared after sample collection were detected by WB. The oxidative stress–associated protein nuclear factor–erythroid 2-related factor-2 (Nrf-2; ab92946, Abcam), lipid peroxidation–associated protein malondialdehyde (MDA; ab27642, Abcam), mitochondrial injury–associated protein cytochrome c oxidase IV (COXIV; ab202554, Abcam), heat shock protein 60 (HSP60; ab190828, Abcam), system Xc−–associated protein solute carrier family 7, member 11 (SLC7A11; ab175186, Abcam), glutathione peroxidase-4 (GPX4; ET1706-45, HUABIO), ferroptosis-associated protein ferroportin-1 (FPN1; ab239511, Abcam), and divalent metal transporter 1 (DMT1; ab157208, Abcam) were used, with β-actin (AF7018, Affinity, England) as the control. Goat anti-rabbit IgG (Biosharp, China) was used as the secondary antibody. A western blotting ECL kit (ZETA-Life, San Francisco, CA, USA) was used. After the detection step, protein bands were observed and photographed using an automated chemiluminescence image analysis system (Tanon, Shanghai, China), and various parameters were quantified using ImageJ software (National Institutes of Health, DC, USA). The procedure was repeated three times before statistical analysis using SPSS 26.0.
Et1706 45
Manufactured by Huabio
Sourced in United Kingdom, China
The ET1706-45 is a laboratory equipment designed for general laboratory use. It is a multi-functional device that can perform various tasks in a research or analytical setting. The core function of this product is to provide a reliable and versatile solution for laboratory applications. No further details on the intended use or specific features are provided, as a concise and unbiased description is required.
Automatically generated - may contain errors
Lab products found in correlation
Ab190828, by Abcam (1 mentions)
Ab239511, by Abcam (1 mentions)
Ab157208, by Abcam (1 mentions)
Ab27642, by Abcam (1 mentions)
Ab202554, by Abcam (1 mentions)
Ab92946, by Abcam (1 mentions)
Ab175186, by Abcam (1 mentions)
Goat anti rabbit igg, by Biosharp (1 mentions)
Af7018, by Affinity Biosciences (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Ab50967, by Abcam (1 mentions)
Ab68477, by Abcam (1 mentions)
A0216, by Beyotime (1 mentions)
Sc 515701, by Santa Cruz Biotechnology (1 mentions)
Er31210, by Huabio (1 mentions)
Hatf00010, by Merck Group (1 mentions)
A0208, by Beyotime (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ab228402, by Abcam (1 mentions)
Meilunbio fg super sensitive ecl luminescence reagent, by Meilun (1 mentions)
7 protocols using et1706 45
1
Protein Expression Analysis of Oxidative Stress
2
Western Blot Analysis of Cardiac Proteins
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Heart tissues and H9c2 cells were lysed via cell lysis buffer, followed by centrifugation at 12,000 rpm for 10 min at 4 °C to obtain the supernatants. A BCA Protein Assay Kit (PC0020, Solarbio) was used to assess the protein concentration in the histiocyte lysates. The corresponding protein samples were added to each well for 10 % or 12 % SDS‒PAGE and separated at 80 or 120 V. Then, the protein was transferred to a nitrocellulose membrane (#HATF00010, Millipore) with a current of 300 mA for 60–90 min. Subsequently, 5 % nonfat milk was used to block the NC membranes at room temperature for 1.5 h, followed by incubation with primary antibodies against ANP (sc-515701, Santa Cruz Biotechnology), COL-1 (sc293182, Santa Cruz Biotechnology), P2X7R (ER1919-21, Huabio), TGF-β (ER31210, Huabio), HuR (ET1705-81, Huabio), GPX4 (ET1706-45, Huabio), β-MYHC (ab50967, Abcam), MMP9 (ab228402, Abcam), and HO-1 (ab68477, Abcam) overnight at 4 °C. The following day, the membranes were incubated with HRP-labeled goat anti-rabbit or anti-mouse IgG (A0208 or A0216, Beyotime) at room temperature for 1 h. Meilunbio® fg supersensitive ECL luminescence reagent (MA0186, Meilunbio) was used to detect protein bands, and ImageJ analysis software was used for protein quantification.
3
Quantifying GPX4 and Neuron Coexpression
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The paraffin sections of mice brains were prepared as above. After antigen retrieval, the tissue sections were blocked with blocking buffer, then incubated with the primary antibody Anti-GPX4 (ET1706-45, HUABIO, China) and Anti-NeuN (66836-1-Ig, Proteintech, China), or Anti-CD8 (GB13429, Servicebio, China) and Anti-CD3 (60181-1-Ig, Proteintech, China) overnight at 4 °C, and with the secondary antibody for 1 h at room temperature, including Cy3 conjugated Goat Anti-Rabbit IgG (GB21303, Servicebio, China) and FITC conjugated Goat Anti-Moue IgG (GB22301, Servicebio, China). The nuclei were stained with DAPI staining solution (G1021, Servicebio, China) for 10 min. Finally, the slides were mounted in Antifade (G1401, Servicebio, China) and examined using digital slide scanning (Pannoramic DESK, P-MIDI, P25), and analyzed using CaseViewer 2.4 (3DHISTECH). The relative expression of GPX4 and NeuN was calculated using fluorescent density, analyzed through ImageJ software, and then standardized to DAPI count.
4
Histopathological and Immunohistochemical Analysis of Kidney Tissue
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Following excision, the kidney was immersed in 4% paraformaldehyde for 2 days at 4°C. Tissues were then dehydrated stepwise in ethanol, cleared with dehydrated xylene, embedded in paraffin, sliced into 4-μm-thick sections, and were deposited on glass slides. Hematoxylin and eosin (H&E) staining was used to obtain the pathological condition. The histological slides of the kidney were evaluated for semiquantitative analysis, as Raij L et al. described previously (Raij L et al., 1984). Masson’s trichrome staining was used to detect the collagen fibers in the kidney. Immunohistochemistry was employed with an anti-GPx4 antibody (1:200, Cat#ET1706-45, HUABIO) and was utilized, according to the manufacturer’s instructions. The intensities of GPx4 in the photos were detected by ImageJ software.
5
Ferroptosis Pathway Regulation Assay
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Reagents: ferrostatin‐1 (CAS:347174–05‐4) and erastin (CAS:571203–78‐6) were all purchased from MedChemExpress (shanghai; China). SBFI26 (CAS:1541207–06‐0) was purchased from GLPBIO Technology (American). Antibodies: Primary antibodies were used as following: anti‐β‐actin antibody (1:1000, mAbcam 8226, Abcam), anti‐ALOX5 (1:500–1:1000, R1512‐14, HuaBio), anti‐ALOX12 (1:2000, AP8877B Abcepta Biotech), anti‐NFE2L2 (1:1000–1:2000, R1312‐8, HuaBio), anti‐GPX4 (1:500–1:2000, ET1706‐45, HuaBio), anti‐HO‐1 (1:1000, ET1604‐45, HuaBio), anti‐ATF4 (1:500–1:2000, ET1612‐37, HuaBio), secondary antibody was purchased from Beyotime (Shanghai, China).
6
Photothermal Therapy of Tumor-Bearing Mice
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Once the tumor sizes reached approximately 100 mm3, the mice were randomly assigned to five groups (each consisting of 5 mice) and subjected to the following treatments: (1) PBS + NIR; (2) Fin56 + NIR; (3) FSR-Fin56; and (4) FSR-Fin56 + NIR. At day 0, 4, 8, and 12, 150 µL of various treatment agents were injected into the tail vein of the tumor-bearing mice. The dose of Fin56 at each injection was 7 mg kg−1 and the pH of PBS was 7.4. At 12 hours post-injection, the tumor site underwent 808 nm NIR laser irradiation (1.0 W/cm2, 6 minutes). Throughout the therapy period, the tumor volume and body weight of the mice were monitored every alternate day. After 16 days of treatment, blood samples were collected, and the tumors along with major organs (heart, liver, lung, spleen, and kidney) were fixed in 4% paraformaldehyde for subsequent experiments. Serum samples were obtained by centrifugation for hepatic and renal function analyses. The main organs and tumor tissues were sectioned for H&E (Solarbio, G1121) and immunofluorescence staining. Antibodies utilized included Ki67 (Affinity, AF0198, 1:200) and GPX4 (Huabio, ET1706-45, 1:100). Fluorescence-conjugated secondary antibodies included donkey anti-rabbit IgG (H + L) FITC (Alexa Fluor 488) (Invitrogen, A-212206; 1:500) and donkey anti-rabbit IgG (H + L) TRITC (Invitrogen, A16026; 1:500).
7
Protein Expression Analysis in Kidney Cells
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Kidney tissues and HK-2 cells were placed on ice and lysed by adding RIPA lysis solution, PMSF and phosphatase inhibitor (all from Servicebio, Wuhan, China) for 30 min. After the protein concentration was determined by BCA method (Beyotime Biotechnology, Shanghai, China), 20 μg of protein was taken for SDS-PAGE electrophoresis, electrotransferred to PVDF membrane, put into TBST containing 0.1% Tween-20 and 5% skim milk for 1 h. The primary antibodies against these targets, YAP (14074, CST, Danvers, MA, USA), ACSL4 (ET7111-43, Huabio, Hangzhou, China), Collagen Ⅰ (ab34710, Abcam, Cambridge, UK), fibronectin (ab2413, Abcam, Cambridge, UK), α-SMA (GB111364, ServiceBio, Wuhan, China), P53 (ab26, Abcam, Cambridge, UK), GPX4 (ET1706-45, Huabio, Hangzhou, China), SLC7A11 (HA600098, Huabio, Hangzhou, China), GAPDH (GB15002, ServiceBio, Wuhan, China), Histone H3 (GB11102, ServiceBio, Wuhan, China), and incubated at 4 °C overnight. The secondary antibodies containing goat anti-mouse/rabbit IgG polymer (SA00001-1, SA0001-0, proteinTech, Wuhan, China) were added and incubated at 37 °C for 1 h. After washing with TBST, Chemi Doc imaging system (Bio-Rad, Hercules, CA, USA) was used to detect protein blots, and Image J software, version 1.52a (NIH, Bethesda, USA) for semi-quantitative analysis.
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