Liver tissues or harvested cells, as indicated, were lysed with RIPA buffer complemented with protease inhibitors. The protein concentration was evaluated through the BCA protein assay kit. In brief, a total of 30 μg proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes. Primary antibodies used were anti-xCT (ab37185, Abcam, 1:1000), anti-GPx4 (ab125066, Abcam, 1:1000), anti-ACSL4 (DF12141, Affinity, 1:1000), anti-p53 (2524, CST, 1:1000), anti-FTH1 (DF6278, Affinity, 1:1000), anti-Nrf2 (12721, CST, 1:1000), anti-LaminB (13435, CST, 1:1000), anti-β-actin (3700, CST, 1:1000), and anti-GAPDH (abs100005, Absin, 1:1000). Second antibodies were peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (1:5000) (Zhongshan Golden Bridge Biotechnology, Beijing, China). To visualize the proteomic bands, an enhanced western luminescent detection kit (Vigorous Biotechnology, Beijing, China) was employed. The densitometry results were quantitatively analyzed by using the Image J software, with β-actin, GAPDH, or LaminB bands being normalized/internal controls as appropriate.
Df12141
Manufactured by Affinity Biosciences
Sourced in China, United States
The DF12141 is a laboratory centrifuge designed for general-purpose applications. It has a maximum speed of 12,000 RPM and can accommodate a variety of sample tube sizes. The centrifuge is capable of separating components of a liquid or solid mixture based on their density differences.
Automatically generated - may contain errors
Lab products found in correlation
Df6278, by Affinity Biosciences (3 mentions)
Df6701, by Affinity Biosciences (3 mentions)
Ab125066, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ab37185, by Abcam (1 mentions)
Scope a1, by Zeiss (1 mentions)
Immunostaining permeabilization buffer with triton x 100, by Beyotime (1 mentions)
Immunol staining blocking buffer, by Beyotime (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Bm5231, by Boster Bio (1 mentions)
Bm5318, by Boster Bio (1 mentions)
A6864, by ABclonal (1 mentions)
β actin, by ABclonal (1 mentions)
Mab3249, by R&D Systems (1 mentions)
Dihydroethidium, by Beyotime (1 mentions)
Af1032, by ABclonal (1 mentions)
S0011, by Affinity Biosciences (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Bs 10411r, by Bioss Antibodies (1 mentions)
Bca kit, by CWBIO (1 mentions)
9 protocols using df12141
1
Protein Expression Analysis in Oxidative Stress
2
Immunofluorescence Staining of Brain Sections
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Immunofluorescence staining was performed as previously described [35 (link)]. Frozen brain sections (7 μm) or cultured cells on coverslips were fixed in 4% paraformaldehyde for 10 min. Then, they were treated with Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime, Shanghai, China) for 30 min and were blocked with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 60 min. These samples were incubated with specific primary antibodies for ACSL4 (Affinity, DF12141, 1 : 200), FTH (Affinity, DF6278,1 : 200), GPX4 (Affinity, DF6701,1 : 200), NeuN (MilliporeSigma, MAB377X, 1 : 200), 8-hydroxyguanosine (8-OHdG) (Bioss, bs-1278R, 1 : 100), and SIRT1 (Santa Cruz, sc-74465, 1 : 50) in Universal Antibody Diluent (New Cell & Molecular, Suzhou, China) at 4°C overnight. Then, the samples were slowly washed three times with phosphate-buffered saline with 0.5% Tween-20 (PBST) and incubated with corresponding secondary antibodies (Cy™3-conjugated goat antirabbit IgG or goat antimouse IgG, 1 : 200) for 1 h at room temperature (RT). After washing 3 times with PBST, the samples were counterstained with 4,6-diamidino-2-phenylindole (DAPI, MilliporeSigma, 1 : 2000) for 10 min at RT. Fluorescence was visualized by a fluorescence microscope (ZEISS, Scope A1, Germany).
3
Ferroptosis-related Protein Detection
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WB was used to determine protein expression levels as previously mentioned.27 We detected ferroptosis‐related protein expressions using anti‐GPX4 antibody (BM5231, Boster, Wuhan, China), anti‐SLC7A11 antibody (BM5318, Boster, Wuhan, China), anti‐ACSL4 antibody (DF12141, Affinity Biosciences, Changzhou, China), and anti‐ALOX15 antibody (A6864, ABclonal, Wuhan, China). β‐Actin (AC026, ABclonal, Wuhan, China) was used as a loading control.
4
Immunodetection of Lipid Metabolism Proteins
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The paraffin kidney sections were applied in immunofluorescence and immunohistochemistry staining to determining the expression levels of various proteins. Primary antibodies for acyl-CoA synthetase long chain family member 4 (ACSL4) (1:200, DF12141, Affinity), glutathione peroxidase 4 (GPX4) (1:200, DF6701, Affinity), 4-hydroxynonenal (4-HNE) (1;100, MAB3249, R&D Systems). Next, the slides were exposed to DAB-labeled secondary antibodies, washed with deionized distilled water, and ultimately observed by light microscopy.
5
Oxidative Stress Evaluation in Corpus Cavernosum
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According to the manufacturer's protocol, superoxide anion and mitochondrial superoxide in the corpus cavernosum were detected with dihydroethidium (Beyotime Biotechnology, China) and MitoSOX (Invitrogen, USA). DAPI was used to stain the cell nuclei.
For immunofluorescence staining, penile sections were incubated with primary antibodies against SMA (1 : 200, Affinity Biosciences, AF1032), GPX4 (1 : 200, ABclonal, A1933), and ACSL4 (1 : 300, Affinity Biosciences, DF12141). Secondary antibodies included DyLight 488- (1 : 300, Affinity Biosciences, S0008), 594- (1 : 300, Affinity Biosciences, S0006), and CY3- (1 : 300, Affinity Biosciences, S0011) conjugated antibodies. Nuclei were stained with DAPI. Images were acquired using a fluorescence microscope (Nikon, Japan) or a confocal laser scanning microscope (Zeiss LSM 880, Germany).
Masson's trichrome staining was performed according to the standard protocol [30 (link)]. The ratio between smooth muscle and collagen in the corpus cavernosum reflects fibrosis of the corpus cavernosum. ImageJ 1.46 (National Institutes of Health, USA) was used for quantitative analysis of the images.
For immunofluorescence staining, penile sections were incubated with primary antibodies against SMA (1 : 200, Affinity Biosciences, AF1032), GPX4 (1 : 200, ABclonal, A1933), and ACSL4 (1 : 300, Affinity Biosciences, DF12141). Secondary antibodies included DyLight 488- (1 : 300, Affinity Biosciences, S0008), 594- (1 : 300, Affinity Biosciences, S0006), and CY3- (1 : 300, Affinity Biosciences, S0011) conjugated antibodies. Nuclei were stained with DAPI. Images were acquired using a fluorescence microscope (Nikon, Japan) or a confocal laser scanning microscope (Zeiss LSM 880, Germany).
Masson's trichrome staining was performed according to the standard protocol [30 (link)]. The ratio between smooth muscle and collagen in the corpus cavernosum reflects fibrosis of the corpus cavernosum. ImageJ 1.46 (National Institutes of Health, USA) was used for quantitative analysis of the images.
6
Western Blot Protein Quantification and Analysis
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The BCA kit (Cwbio, Jiangsu, China) was utilized to quantify the protein isolated from cells and tissues, followed by being separated with the 12% SDS-PAGE. The separated protein was transferred from the gel to the PVDF membrane, which was further introduced with 5% skim milk. Then, the membrane was introduced with the primary antibody against P-GP (1:1000, 22336-1-AP, Proteintech, USA), ERCC1 (1:1000, DF7255, Affinity, USA), NOX1 (1:1000, 17772-1-AP, Proteintech, USA), COX2 (1:1000, bs-10411R, Bioss, USA), GPX4 (1:1000, 67763-1-Ig, Proteintech, USA), FTH1 (1:1000, DF6278, Affinity, USA), ACSL4 (1:1000, DF12141, Affinity, USA), FASN (1:1000, 66591-1-Ig, Proteintech, USA), USP14 (1:1000, sc-515,812, Santa Cruz, USA), and β-actin (1:1000, HC201, TransGen Biotech, China). The second antibody (1:2000, GB23303, Servicebio, China) was subsequently added to be incubated for 90 min. Finally, ECL reagent was added to expose the bands, which were further quantified with the ImageJ software.
7
Western Blot Analysis of Hepatocyte Proteins
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Protein lysates, obtained from hepatocytes and liver specimens, were electrophoresed on 4–12% precast bis–tris gels (Genscript, Nanjing, China) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked using protein-free rapid blocking buffer (EpiZyme, Shanghai, China) and incubated with primary antibodies overnight at 4 °C. Antibodies used were as follows: Med1 (ab243893, Abcam), Nrf2 (D1Z9C) (12721S, CST), HO-1 (R24541, Zen BioScience), ACSL4 (DF12141, Affinity Biosciences), TfR1 (ab269513, Abcam), SLC7A11 (ab175186, Abcam), GPX4 (ab125066, Abcam), NQO1(DF6437, Affinity Biosciences) and β-Tubulin (66,240–1-Ig, Proteintech).
8
Western Blot Analysis of Protein Markers
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The colon tissues or cell lines were disintegrated with cold RIPA buffer (Beyotime,P0013B) supplemented with protease inhibitors (Solarbio, China) on ice for 30 min. The supernatant was collected after adequate homogenization and centrifugation. The proteins were loaded onto an SDS-polyacrylamide gel electrophoresis system with 6 μl of the initial protein amount. The exact amount of different groups was adjusted according to the internal control. The size-separated proteins were blotted onto polyvinylidene fluoride (PVDF) membranes (Invitrogen, USA). After blocking with 10% bovine serum albumin, the membranes were cut into bands and incubated with primary β-actin antibody (1:1000, ab8226, Abcam), anti-GPX4 antibody (1:1000, #DF6701, Affinity Biosciences), anti-ACSL4 antibody (1:1000, #DF12141, Affinity Biosciences), anti-HIF-2-alpha antibody (1:1000, #DF2928, Affinity Biosciences) and anti-DMT1 antibody (1:1000, bs-3577R, Bioss) overnight at 4 °C. On the following day, the bands were incubated with HRP-linked anti-mouse IgG (1:5000, Cell Signaling Technology) or HRP-linked anti-rabbit (1:5000, Cell Signaling Technology) for 1 h at room temperature. The immuno-reactive bands were detected by enhanced chemiluminescence and analyzed by Image J software (Image J 1.52).
9
Western Blot Analysis of GPX4, ACSL4 in Molm-13 Cells
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Molm-13 cells were collected and lysed with RIPA buffer (BL504A, Biosharp, China) for 30 min on ice. Then, the protein (50 μg) from each sample was separated by 10% tris-acrylamide gel electrophoresis and transferred onto the PVDF membrane. After blocking with 5% skim milk for 1 h at room temperature, primary antibodies against GPX4 (DF6701, Affinity, United States), ACSL4 (DF12141, Affinity, United States), and GAPDH (AF7021, Affinity, United States) were used and incubated at 4°C overnight on a rotary shaker. After washing with TBST 5 times, the membrane was probed with goat anti-rabbit IgG highly cross-adsorbed secondary antibody (1:10,000) for 1 h at room temperature. Then, the membrane was washed 3 times with TBST and developed with an ECL reagent.
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