Total protein was extracted with RIPA buffer (Beyotime, China). Next, the concentration of these samples was determined with the BCA method (Beyotime, China). Then, these proteins were separated by the 10% SDS-PAGE gel (Beyotime, China). After that, these proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, USA). These membranes were blocked with 5% defatted milk at room temperature for 2 h and incubated with the primary antibodies at 4°C overnight. The primary antibodies used in this research were GPX-4 (ab125066, Abcam), solute carrier family 7 member 11 (SLC7A11; ab37185, Abcam), solute carrier family 3 member 2 (SLC3A2; sc-390154, Santa Cruz), arachidonate-5-lipoxygenase (ALOX5; ab169755, Abcam), autophagy-related 5 (ATG5; ab108327, Abcam), ATG7 (ab133528, Abcam), nuclear receptor coactivator 4 (NCOA4; H00008031-M04, Novus), transferrin receptor (TFR1, ab84036, Abcam), divalent metal transporter 1 (DMT-1; ab222895, Abcam), and GAPDH (ab9485, Abcam). On the second day, these membranes were washed with PBST and incubated with the secondary antibody (Goat anti-rabbit IgG, ab150077, Abcam) for 2 hours. Finally, the bands were developed with enhanced chemiluminescence (ECL) substrates (Millipore, USA).
Ab169755
Manufactured by Abcam
Sourced in United States
Ab169755 is a primary antibody used for research purposes. It is a polyclonal antibody produced in rabbit and recognizes a specific target protein. The core function of this product is to bind and detect the target protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab125066, by Abcam (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab108327, by Abcam (1 mentions)
Ab84036, by Abcam (1 mentions)
Ab37185, by Abcam (1 mentions)
Ab133528, by Abcam (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Bca method, by Beyotime (1 mentions)
Ab211506, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Pv 9000, by ZSGB-BIO (1 mentions)
Ab64693, by Abcam (1 mentions)
Universal hier antigen retrieval reagent, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
Ab175186, by Abcam (1 mentions)
Ab178846, by Abcam (1 mentions)
8 protocols using ab169755
1
Western Blot Analysis of Ferroptosis-Related Proteins
2
Immunohistochemical Analysis of ALOX5, ALOX12, and CISD1 in Pancreatic Cancer
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Immunohistochemistry was performed by two-step method according to the instructions (PV-9000; ZSGB-BIO, Beijing, China). Pancreatic cancer samples were fixed in 10% formalin, embedded in paraffin, and processed into 5-µm sequential sections. The samples were de-waxed with ethanol and blocked to inhibit the endogenous peroxidase activity. After this, samples were heated in a microwave for antigen retrieval, cooled to room temperature, and blocked using goat serum for 30 min at 37 °C. The samples were incubated overnight at 4 °C with rabbit anti-ALOX5 (ab169755), anti-ALOX12 (ab211506), and anti-CISD1 (ab203096) (Abcam, USA) (1:200), followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody (PV-9000; ZSGB-BIO, Beijing, China) at 37 °C for 30 min. The samples were then stained with 3,3′-Diaminobenzidine (DAB). Cell nuclei were stained blue with hematoxylin. The sections were then dehydrated, cleared with xylene, and mounted. ALOX5, ALOX12, and CISD1 expressions were determined by immunohistochemistry (IHC) using the streptavidin peroxidase method, with adjacent tissues serving as the controls. The experimental procedure was performed as per the manufacturer’s instructions. Image-Pro Plus 6.0 Software (Media Cybernetics, USA) was used to analyze protein expression and perform statistical analysis of the results obtained by IHC.
3
Sequential Immunostaining of Murine Lung
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Sequential immunostaining was performed on 3-μm-thick Formalin-Fixed Paraffin-Embedded (FFPE) murine left lung sections as previously described80 . Briefly, heat mediated antigen retrieval was performed in Universal HIER antigen retrieval reagent (Abcam, #ab208572). Antibody elution was performed between each staining cycle. Primary antibodies used were: rabbit anti-5-Lipoxygenase/5-LO (1:500, Abcam #ab169755), rabbit anti-Myeloperoxidase (1:1000, Abcam #ab208670), rabbit anti-IBA1 (1:1000, VWR #100369–764), rabbit anti-iNOS (1:250, Abcam #ab239990), and rabbit anti-CD206 (1:250, Abcam #ab64693). Secondary antibodies used were: anti-rabbit 555 (1:500, Cell Signaling #4413 S) and anti-rabbit 647 (1:1000, Cell Signaling #4414 S).
Acquired images were processed using FIJI and the FIJI plugin HyperStackReg V5.681 (link) (and Ved Sharma. 2018, December 13 Zenodo. 10.5281/zenodo.2252521). Autofluorescence acquired in nonrelevant channels was substracted as appropriate. Quantitation was performed using Ilastik (v1.3.3post3)82 (link) and CellProfiler (4.1.3)83 (link).
Acquired images were processed using FIJI and the FIJI plugin HyperStackReg V5.681 (link) (and Ved Sharma. 2018, December 13 Zenodo. 10.5281/zenodo.2252521). Autofluorescence acquired in nonrelevant channels was substracted as appropriate. Quantitation was performed using Ilastik (v1.3.3post3)82 (link) and CellProfiler (4.1.3)83 (link).
4
Quantifying Ferroptosis-Related Proteins in Spinal Cord Injury
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0.5-cm spinal cord tissues around the lesion center were harvested with PBS perfusion and homogenized on ice in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) to quantify ferroptosis-related proteins. 20 μg of the protein in each well was loaded on 12% SDS-PAGE and transferred onto a PDVF membrane, which was blocked with 5% skimmed milk at room temperature for 1.5 h followed by overnight incubation with primary antibodies against glutathione peroxidase 4 (GPX4, 1:5000, ab125066, and Abcam), System Xc− light chain (xCT, 1:2000, ab175186, and Abcam), Acyl-CoA synthetase long-chain family member 4 (ACSL4, 1:5000, ab155282, and Abcam), 5-lipoxygenase (5-LOX, 1:1000, ab169755, and Abcam), and IBA-1 (1:1000, ab178846, and Abcam) at 4°C. After rinsing with Tris-buffered saline and Tween (TBST), the membrane was incubated with secondary antibody (Anti-mouse IgG, HRP-linked Antibody, 1:1000, 7076, CST; Anti-rabbit IgG, HRP-linked antibody, 1:1000, 7074, CST; Danvers, MA, United States) at room temperature for 1 h. Chemiluminescence was detected and quantified using ImageJ software. β-actin (1:5000, sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, United States) and GAPDH (1:1000, ab8245, and Abcam) were used as the internal standard. The mean and SD of relative protein levels across at least three independent experiments were used for statistical comparisons.
5
Protein Isolation and Analysis in Lung Tissue
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Total proteins were isolated from right lung tissue (50 µg) and cells (5×106) using RIPA buffer (#89900; Thermo Scientific, Worcester, MA, USA) containing a protease inhibitor cocktail (#P2714; Sigma) and phosphatase inhibitor cocktail (#04906845001; Roche Applied Science, Indianapolis, IN, USA). Homogenates were centrifuged at 2,834 g at 4 °C for 20 min before the supernatant was collected. The total protein concentrations were determined by bicinchoninic acid assay (#23227, PierceTM Biotechnology, Rockford, IL, USA). Standard SDS-PAGE technique (27 (link)) was performed on equal amounts of proteins, with antibodies of p-STAT5 (ab32364; 1:500), RORγt (ab135669; 1:500), FOXP3 (ab215206; 1:500), JNK (ab112501; 1:500), p-JNK (ab4821; 1:500), arachidonate 5-lipoxygenase (Alox5; ab169755; 1:500), activator protein 1 (AP-1, ab21981; 1:500), and GAPDH (ab181602; 1:500) obtained from Abcam. The band intensity was measured by ImageJ2× 2.1.4.7 (Wayne Rasband, National Institutes of Health, USA).
6
MDCK Cell Protein Analysis
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Whole cell lysates of MDCK cells were prepared by homogenization in RIPA buffer. The whole cell lysate of MDCK cells was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 5-LOX and actin were analyzed by immunoblotting using an anti-5-LOX monoclonal antibody (ab169755, dilution 1/1,000; Abcam, Cambridge, UK) and anti-actin antibody (MAB1501R, dilution 1/2,000; Chemicon International, Temecula, CA).
7
Visualizing 5-LOX, CysLTR1 and Aggregates
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5-LOX, CysLTR1 and aggregated proteins (aggresomes and related inclusion bodies) were visualized in fixed ARPE-19 cells using IF analysis as recently described27 (link). 5-LOX and CysLTR1 were labeled using the anti-5-LOX antibody (1:100; ab169755, Abcam) and anti-CysLTR1 antibody (1:100; ab151484, Abcam), respectively. The primary antibodies were labeled using a donkey anti-rabbit antibody conjugated to Alexa Fluor 488 (1:1000, A-21206, Thermo Fisher Scientific) or Alexa Fluor 647 (1:1000; A-31573, Thermo Fisher Scientific). Aggregated proteins were visualized using Proteostat (1:500), which was added during the secondary antibody incubation step for 1 h at RT. To discriminate between specific and nonspecific fluorescence signals, secondary-antibody-only controls were used.
8
Western Blot Analysis of Retinal Proteins
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ARPE-19 cells (n = 3) and mouse RPE/choroid tissue of one eyeball (n = 3) were lysed in M-PER and T-PER (both from Thermo Fisher), respectively. All samples were mixed with a protease/phosphatase inhibitor cocktail (Abcam), and protein concentration was measured using Coomassie protein assay. Protein samples (10-15 μg) were prepared with 4x Laemmli Sample Buffer with 1.25% (v/v) 2-mercaptoethanol and denatured at 90°C for 5 min. Equal amounts of protein were separated on an SDS-PAGE gel and subsequently transferred to PVDF membranes with a wet transfer system (Hoefer, Holliston, MA, USA). The PVDF membrane was then blocked with Protein Block (Abcam). Next, the membranes were incubated with primary antibodies for 5-LOX (ab169755, Abcam), GPx4 (ab125066, Abcam), succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA, #11998, Cell Signaling, Danvers, MA, USA), human xCT/SLC7A11 (#12691, Cell Signaling), and mouse xCT (NB300-318, Novus, Centennial, CO, USA), diluted in TBST with 5% (w/v) BSA overnight at 4°C. The membranes were then incubated with HRP-linked secondary antibodies for 1 h at room temperature (RT). The protein bands were visualized with HRP substrate reagent (Thermo Fisher) and detected with an ECL imaging system (Bio-Rad, Hercules, CA, USA). The relative expression of target protein was obtained after normalizing to β-actin (ab8227, Abcam) expression.
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