Hippocampal tissue was used for western blotting analysis 24 h after LPS microinjection (n = 6). Briefly, 50 μg of protein was separated on a 4–12% SDS-PAGE gel and transferred to a PVDF membrane. The primary antibodies were anti-Gpx4 (1:100; Senta Cruz, TX, USA), anti-MtFt (1: 250; Gene Tex, CA, USA), anti-Fth (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-xCT (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-TF (1:1000; Abcam, Cambridge, UK), anti-GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA). The membranes were incubated with anti-rabbit or anti-mouse fluorophore-conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) for 1.5 h, and bands were detected using an Odyssey scanner (LI-COR, Lincoln, NE, USA). The images were analyzed using HIN ImageJ software.
Anti fth
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Fth is a primary antibody that recognizes the Ferritin Heavy Chain (Fth) protein. Fth is a subunit of the ferritin protein complex, which is responsible for the storage and regulation of iron in cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (2 mentions)
Odyssey scanner, by LI COR (1 mentions)
Anti tf, by Abcam (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti xct, by Cell Signaling Technology (1 mentions)
Anti protease and anti phosphatase cocktails, by Roche (1 mentions)
Clarity ecl kit, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Anti lc3 1 2, by Cell Signaling Technology (1 mentions)
Anti ripk3, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti xct, by Abcam (1 mentions)
Anti gpx4, by Cell Signaling Technology (1 mentions)
3 protocols using anti fth
1
Western Blot Analysis of Hippocampal Proteins
2
Western Blot Analysis of Ferritin
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BM cells from BM Fth−/− or BM Fth+/+ were isolated. Briefly, cells were lysed in lysis buffer (1% Triton X-100, 150mM NaCl, 20mM HEPES pH7.5, 10% glycerol, 1mM EDTA, supplemented with anti-protease and anti-phosphatase cocktails, Roche) and protein concentration was determined using BCA assay (Pierce). 20 μg of protein was resolved by SDS-PAGE and transferred onto PVDF membranes (Biorad). Membranes were blocked and incubated overnight at 4°C with gentle agitation with anti-Fth (1:1000, Cell Signaling Technologies) or actin (1:10 000, Sigma-Adrich). Then primary antibodies were conjugated to secondary HRP-conjugated antibodies and the signal was detected using Clarity ECL kit (Biorad) and acquired on Chemidoc MP System (Biorad).
3
Protein Expression Analysis of Heart Tissue
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The collected mice hearts and NRCs were lysed in radioimmunoprecipitation assay (RIPA) buffer. Bicinchoninic acid protein assay kits (Thermo Scientific) were used to measure the protein concentrations. Protein was isolated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred into a polyvinylidene fluoride membrane and incubated at room temperature for 2 h with a 5% non‐fat milk-blocking buffer. Afterward, membranes were incubated with the primary antibodies at 4°C overnight, including anti-Clearved Caspase-3 (1:1000; cat#: 9664, Cell Signaling Technology), anti-RIPK3 (1:1000; cat#: 10188, Cell Signaling Technology), anti-LC3 I/II (1:1000; cat#: 4108, Cell Signaling Technology), anti‐Gpx4 (1:1000; cat#: 59735, Cell Signaling Technology), anti‐xCT (1:1000; cat#: ab300667, Abcam), anti‐FTH (1:1000; cat#: 4393, Cell Signaling Technology), anti‐Nrf2 (1:1000; cat#: ab62352, Abcam), and anti‐β actin (1:10000, cat#: 4970, Cell Signaling Technology). The membranes were incubated at room temperature for 2 h with secondary antibodies (1:10000, Boster, Shanghai, China) and visualized by ECL kits (Engreen, Beijing, China). The protein expression was quantified by ImageJ software and was normalized to control.
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