Antibodies against the following proteins were used for western blot analyses: SLC7A11 (ab175186 and ab216876; Abcam, Cambridge, MA, USA), hypoxia-inducible factor 1-alpha (HIF1α; 66730-1-Ig; Proteintech, Rosemont, IL, USA), FASN (A0461; ABclonal Technology, Woburn, MA, USA), and β-actin (AC026; ABclonal Technology).
Ab216876
Manufactured by Abcam
Sourced in United States
Ab216876 is a laboratory equipment product. It is designed for a specific function within a laboratory setting. A detailed description of the product's core function is not available without the risk of unbiased interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab125066, by Abcam (4 mentions)
Ab32443, by Abcam (2 mentions)
Ab108928, by Abcam (2 mentions)
Ab109376, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab175186, by Abcam (1 mentions)
β actin, by ABclonal (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab53154, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ab6728, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Ab91654, by Abcam (1 mentions)
Ifn γ 300 02, by Thermo Fisher Scientific (1 mentions)
Ferrostatin 1, by Selleck Chemicals (1 mentions)
Ab8227, by Abcam (1 mentions)
8 protocols using ab216876
1
Protein Expression Analysis by Western Blot
2
Protein Expression Analysis in Cell Lysates
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Tissue homogenates and cells were treated with RIPA lysates (Beyotime, China), and supernatants containing proteins were collected by centrifugation. Protein content was assessed by BCA detection kit (EMD Millipore). SDS-PAGE was performed with the same amount of protein samples in each lane, and then the isolated proteins were electrotransferred to PVDF membrane (Millipore). 5% skimmed milk powder was used for blocking the membranes, and then the corresponding primary antibody (eIF5A (1 : 1000, ab32443, Abcam); FANCD2 (1 : 1000, ab108928, Abcam); SLC7A11 (1 : 1000, ab216876, Abcam); HSPB1 (1 : 1000, ab109376, Abcam); Bax (1 : 1000, ab53154, Abcam); Bcl-2 (1 : 1000, ab32124, Abcam); cleaved caspase-3 (1 : 1000, ab32042, Abcam); cytochrome C (1 : 1000, ab133504, Abcam); β-actin (1 : 1000, ab8226, Abcam)) was applied overnight at 4°C. Following that, the membranes were treated for 2 h with the corresponding secondary antibody (Goat Anti-Rabbit IgG H&L (1 : 2000, ab6721, Abcam) and Rabbit Anti-Mouse IgG H&L (1 : 2000, ab6728, Abcam)). The transfer protein on membranes was developed with electrochemiluminescence (ECL, Thermo Fisher Scientific, USA)). Grayscale of the strips was assessed by ImageJ 1.48v software (NIH).
3
Antibodies and Ferroptosis Inducers Protocol
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Antibodies against CRTC3 (ab91654, WB, 1:1000), SLC7A11 (ab216876, WB, 1:1000), GPx4 (ab125066, WB 1:1000, IHC 1:100), and β-actin (ab8227, WB, 1:1000) were purchased from Abcam. Sorafenib (S7397), erastin (S7242), RSL3 (S8155) and Ferrostatin-1 (S7243) were purchased from Selleck Chemicals. PRGL493 (HY-139180) was purchased from MedChemExpress. IFN-γ (300-02) was purchased from PEPROTECH.
4
Immunohistochemical Analysis of Cellular Markers
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Slices were dewaxed and hydrated. Endogenous catalase was removed by H2O2. The slices were blocked for 30 min at 37°C with 5% BSA solution (P0220, Beyotime). Following that, the sections were reacted with primary antibody [eIF5A (1 : 250, ab32443, Abcam); FANCD2 (1 : 100, ab108928, Abcam); SLC7A11 (1 : 500, ab216876, Abcam); HSPB1 (1 : 500, ab109376, Abcam)] overnight at 4°C. The sections were incubated with the secondary antibody for 30 min. Then the slides were treated for 5 min with diaminobenzidine (DAB, Beyotime, China). After restaining for 5 min with hematoxylin, the slices were dehydrated, made transparent, and finally sealed with neutral resin. The results were observed with an optical microscope (Olympus Corporation).
5
Western Blot Analysis of SLC7A11, GPX4
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The tissues and cells were lysed with radioimmunoprecipitation assay lysis buffer (Thermo Fisher, USA). The cell lysates were divided in sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The protein bands were probed with primary antibodies against SLC7A11 (ab216876, Abcam), GPX4 (ab125066, Abcam), and β-actin (ab7817, Abcam) at 4 ℃ overnight, and then incubated with horseradish peroxidase-conjugated secondary antibodies. The protein bands were then visualized after reaction with enhanced chemiluminescence reagent (Millipore, Germany).
6
Protein Extraction and Western Blot Analysis for LUAD Cells
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RIPA lysis buffer (RIPA: PMSF = 100:1) was used to extracted the inner protein of LUAD cells. After measured all densities, the prepared proteins (30 µg/lane) were orderly separated by SDS-PAGE (10% or 12%) and then transferred onto pre-cut PVDF membranes. After blocking in skimmed experimental milk (5%) for 2 h at 37 ℃, the targeted membranes were incubated with preconfigured primary anti-FTH (#4393; 1:600; CST; USA), PTGS2 (#12,282; 1:600; CST; USA), DMTI (#15,083; 1:800; CST; USA), SLC7A11 (ab216876; 1:800; Abcam; USA), GPX4 (ab125066; 1:800; Abcam; USA) and β-actin (#4970; 1:3000; CST; USA) overnight at 4 °C. Next day, these were treated with corresponding secondary antibody. Finally, protein signals on the finished membrane were visualized by enhanced ECL kit (4 A Biotech, China).
7
Protein Expression Analysis by Western Blotting
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Total proteins were extracted using RIPA (Thermo Fisher Scientific), and the protein concentration was estimated through the BCA kit (Beyotime). Proteins were separated using the 10% SDS-page gel and further transferred into a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked and subsequently incubated overnight with antibodies against SLC7A11 (Abcam, 1:1000, ab216876), GPX4 (Abcam, 1:1000, ab125066), FOSL2 (Cell Signaling Technology, 1:1000, #19967), NAMPT (Cell Signaling Technology, 1:1000, #86634), p53 (Abcam, 2 μg/ml, ab26), p-p53 (Abcam, 1:1000, ab33889), and GAPDH (Abcam, 1:2500, ab9485). The membranes were then incubated with the corresponding secondary antibody (Abcam, 1:5000, ab7090) for 60 min following washed with PBST. The bands were visualized using an ECL detection kit (Beyotime).
8
Immunoblotting Analysis of Protein Expression
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Cells and homogenized tissues were lysed in radioimmunoprecipitation assay buffer, and proteins were separated on 4–20% gradient gels. Protein concentration was measured using the Omni-Easy™ Instant BCA Protein Assay Kit (ZJ102, EpiZyme, China). Primary antibodies against CD9 (1:1000, ab236630), CD63 (1:1000, ab134045), CD81 (1:1000, ab79559), Alix (1:1000, ab275377), calnexin (1:1000, ab133615), CitH3 (1:1000, ab5103), PAD4 (1:1000, ab96758), GPX4 (1:1000, ab125066), ACSL4 (1:1000, ab155282), TFR1 (1:1000, ab214039), and SLC7A11 (1:1000, ab216876) were purchased from Abcam (USA). Primary antibodies against H3 (1:1000, 4499S), phosphoinositide-3-kinase (PI3K) (1:1000, 17366S), p-AKT (1:1000, 4060S), and AKT (1:1000, 4685S) were purchased from Cell Signaling Technology (CST, USA). The anti-GAPDH (housekeeping protein) antibody (1:1000, 5174S) was purchased from CST. Densitometry was performed using ImageJ (version 1.47) software. The experiment was performed in triplicate.
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