Ab239583

Total protein was isolated by lysing cells with RIPA lysis buffer supplemented with protease inhibitor cocktail. Protein quantification was done with BCA protein assay kit (PC0020, Solarbio, China). Equal quantities of protein samples were then mixed with loading buffer, heated for 10 min at 100 °C, separated on 10% SDS-PAGE gels, and subsequently transferred to PVDF membranes, which were then blocked with 5% nonfat milk for 1 h, exposed overnight (O/N) to specified antibodies at 4 °C, followed by three 15-min TBST rinses, and further exposure to anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (2° Ab, 1:5000) at room temperature (RT) for 1 h, before visualization of the protein bands with ECL reagent and Amersham Imager 600 (General Electric Company, USA). Finally, the protein bands were quantified with Image J gel analysis software. All experiments were repeated three times. Among the primary antibodies (1° Abs) used were: Rabbit monoclonal anti-TERT (ab191523, Abcam, 1:1000); anti-Nrf2 (ab137550, Abcam, 1:1000); anti-SLC7A11 (ab37185, Abcam, 1:1000); anti-Ferritin (ab75973, Abcam, 1:1000); anti-Ferroportin (ab239583, Abcam, 1:1000); anti-GPX4 (ab125066, Abcam, 1:1000); and anti-β-Actin (4970S, Cell Signaling Tech, 1:1000).

RIPA lysis buffer containing a cocktail (50×) was used to lyse cells. After isolating proteins from homogenates, SDS-PAGE gels (Beyotime) were used for electrophoresis. Proteins were transferred onto a PVDF membrane and incubated in 5% dry non-fat milk. Immunoblotting was performed overnight at 4 °C using primary antibodies against SLC7A11 (26864-1-AP, Proteintech, Rosemont, IL, USA), NFS1 (15370-1-AP, Proteintech), GPX4 (ab125066, abcam, Cambridge, UK), Ferritin heavy chain (FTH, ab75972, abcam), Ferritin light chain (FTL, ab75973, abcam), SLC40A1 (FPN, ab239583, abcam), Mitochondrial ferritin (MTFT, ab124889, abcam), Transferrin receptor (TFR, ab214039, abcam), SLC11A2 (divalent metal transporter 1, DMT1, 20507-1-AP, Proteintech), NRF2 (16396-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech). The primary antibodies were diluted in Primary Antibody Dilution Buffer (Beyotime). Horseradish peroxidase-conjugated secondary antibodies (Proteintech) diluted in 5% FBS were then applied to the membranes for another 1.5 h at room temperature. Chemiluminescent signals were detected by the ChemiDoc imaging system (Tanon, Urumqi, China) after development by enhanced chemiluminescence (Millipore, Burlington, MA, USA).

After the H9C2 cells in different groups were digested, centrifuged and lysed, the total proteins were isolated for detection of protein concentration. 30 μL of protein samples was taken for electrophoresis in 12% SDS-PAGE gel, electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with primary antibodies overnight at 4°C. The corresponding horseradish-peroxidase-labeled IgG (1:5,000; Abcam, Cambridge, UK) was added on the next day for 1 h before ECL color development. The relative quantity of protein expression in different groups was calculated using Image J software. Primary antibodies used in the study were: Anti – Nrf2 (1:1000; ab62352; Abcam); Anti – SLC7A11 (1:1000; ab175186; Abcam); Anti-ferritin heavy chain (FTH1, 1:1000; ab75972; Abcam); Anti – GPX4 (1:1000; ab125066; Abcam); Anti – FPN1 (1:1000; ab239583; Abcam); Anti – NOX1 (1:1000; ab121009; Abcam); Anti-GAPDH (1: 1000; ab8245; Abcam).