The protein level was detected by following the previously described protocol (Lv et al., 2020 (link)). In detail, whole embryos of alas2 or alad mutants and their siblings at 36 hpf were collected for Alas2 and Alad protein level detection using anti-Alas2 (1:2000, GTX127887, GeneTex) or anti-Alad (1:2000, ab59013, Abcam) antibody, respectively. The flow cytometric-sorted 30,000 HSPCs (kdrl+/cmyb+) of control, alas2−/− or alad−/− at 36 hpf were used for detection of Fth1, Gpx4, Slc7a11 and Tfr1b protein levels using anti-Fth1 (1:2000, A19544, ABclonal), anti-Gpx4 (1:1000, A1933, ABclonal), anti-Slc7a11 (1:1000, A15604, ABclonal) and anti-Tfr1b (1:2000, 10084-2-AP, Proteintech) antibodies, respectively.
A19544
Manufactured by ABclonal
Sourced in United States
A19544 is a lab equipment product from ABclonal. It is a device used for the centrifugation of samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
A1933, by ABclonal (2 mentions)
A5865, by ABclonal (2 mentions)
Ab59013, by Abcam (1 mentions)
A2856, by Merck Group (1 mentions)
Rgar001, by Proteintech (1 mentions)
Rgam001, by Proteintech (1 mentions)
Ac004, by ABclonal (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Ferrostatin 1 fer 1, by Selleck Chemicals (1 mentions)
A1253, by ABclonal (1 mentions)
A6826, by ABclonal (1 mentions)
Ab208035, by Abcam (1 mentions)
Ab76572, by Abcam (1 mentions)
A2413, by ABclonal (1 mentions)
P jnk, by Abcam (1 mentions)
A0216, by Beyotime (1 mentions)
A1244, by ABclonal (1 mentions)
A0208, by Beyotime (1 mentions)
Ab85476, by Abcam (1 mentions)
Ab1424, by Abcam (1 mentions)
5 protocols using a19544
1
Proteomic Analysis of Heme Biosynthesis Genes
2
Protein Lysate Preparation and Immunoblotting
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Total protein was lysed from the cells using radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton, 0.1% sodium dodecyl sulphate [SDS], 1 mM ethylenediaminetetraacetic acid [EDTA], 1% sodium deoxycholic acid, 1 mM Na3VO4, and 10 mM NaF) with protease inhibitors and phenylmethylsulfonyl fluoride (PMSF). Equivalent amounts of total cellular lysates were separated using 10% SDS-polyacrylamide gel electrophoresis. Primary antibodies used for immunoblotting included anti-ATG7 (1:1000; A2856, Sigma), and antibodies to FTH1 (1:1000; A19544, ABclonal), β-actin (1:10000; AC004, ABclonal). Horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies (RGAR001 or RGAM001, Proteintech) were used, and signals were determined using Western ECL Substrate. Uncropped data were shown in supplementral material .
3
Dapagliflozin and Ferroptosis Inhibition
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Dapagliflozin was obtained from MCE (Cat. HY-10450). Ferrostatin-1 (Fer-1) was purchased from Selleck Chemicals (Cat# S7243). Drugs were separately dissolved in DMSO and added to the culture medium 12 h before H/R. For in vivo experiments, DAPA was purchased from commercially available dapagliflozin tablets (Forxiga Tab 10 mg, AstraZeneca AB, United States). DAPA was diluted with sterile water for injection (WFI) and given to animals by intragastric administration, as previously shown (Nikolaou et al., 2022 (link)). Primary antibodies against β-tubulin (1:5,000, Cat. AC021), GPX4 (1:1,000, A1933), SLC7A11 (1:1,000,A2413), ACSL4 (1:1,000, A6826), PTGS2 (1:1,000, A1253), and FTH1 (1:1,000, A19544) were purchased from ABclonal (Wuhan, China); FTMT (1:200,aa65-227) was purchased from LifeSpan BioSciences (Seattle, WA, United States); p-ERK (1:1,000,#4370), ERK (1:1,000, #4695), p-P38 (1:1,000, #4511), and P38 (1:1,000, #8690) were purchased from Cell Signaling Technology (Beverly, MA, United States); p-JNK (1:1,000, ab76572)), and JNK (1:1,000, ab208035) were purchased from Abcam (San Francisco, CA).
4
Western Blot Analysis of Cellular Proteins
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Fresh tissue and cells were disrupted with a high-strength RIPA lysis buffer, followed by 30 min of standing and centrifugation at 12,000 rpm at 4°C for 10 min. The protein content of the supernatant was quantified using a bicinchoninic acid (BCA) standard curve. Ten microliters of samples with equal amounts of total protein were added to 12.5% or 7.5% gels and electrophoresed at a stable voltage of 200 V. The gels were then transferred onto a PVDF membrane at a constant current of 400 mA. After blocking with 5% skimmed milk for 2 h, the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-Bax (1:2000, 50599-2-Ig, Proteintech), rabbit anti-Bcl-2 (1:2000, 26593-1-AP, Proteintech), mouse anti-GPX4 (1:2000, 67763-1-Ig, Proteintech), rabbit anti-Nrf2 (1:3000, A1244, ABclonal), rabbit anti-CD71 (1:1000, A5865, ABclonal), rabbit anti-FTH1 (1:1000, A19544, ABclonal), rabbit anti-DMT1 (1:2000, 20507-1-AP, Proteintech), rabbit anti-Fpn1 (1:1000, 26601-1-AP, Proteintech), and mouse anti-β-actin (1:1000, 66009-1-Ig, Proteintech). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:3000, A0208, Beyotime) and goat anti-mouse (1:1000, A0216, Beyotime) antibodies were added for 1 h, followed by enhanced chemiluminescence (ECL) visualization.
5
Western Blot Analysis of Iron Regulators
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Western blot analysis was performed as previously described [24 (link)]. Antibodies in this work were anti-KLF14 (ab85476; Abcam, Cambridge, MA, USA), anti-IRP1 (12406–1-AP, Proteintech, 1:1000), anti-IRP2 (23829–1-AP, Proteintech, 1:1000), anti-TfR1 (A5865, ABclonal, 1:1000), anti-Ferritin H (A19544, ABclonal, 1:1000), anti-SIRT1 (A17307, ABclonal, 1:1000), anti-GAPDH (M20006, Abmart, 1:5000), anti-Flag (M20008, Abmart, 1:5000, 1:1000), anti-HA (ab1424，abcam, 1:1000), anti-Actin (ab8227, abcam, 1:1000).
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