Ab124889

OVCAR3 and SKOV3 cells transfected with shRNA-CREB or negative control were treated with Roflumilast and/or H89, and then the cells were harvested and, according to the manufacturer's instructions, the whole cell protein extracts were resolved on a 10% SDS denatured polyacrylamide gel and were then transferred onto a nitrocellulose membrane, which were blocked in 5% BSA in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated with Anti-GAPDH (#ab8245, Abcam, USA), FtMt (#ab124889, Abcam, USA), CREB (#ab32096, Abcam, USA), p-CREB (#9198, Cell Signaling Technology, USA) antibodies overnight at 4°C. GAPDH was used as the loading control in the Western blotting. After washing with TBST buffer, the blots were then incubated with HRP-conjugated secondary antibody (#BS12478, #BS13278, Bioworld, China) for 1 h at room temperature. After washing with TBST buffer, the blots were visualized using the ECL-Plus reagent (Millipore, Billerica, MA, USA).

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).

HepG2 cells were lysed in lysis buffer (Beyotime, China) containing protease inhibitor. Protein concentration was determined using bicinchoninic acid protein assay kits (Beyotime, China). 20 µg of proteins were loaded on to 10% SDS-PAGE gels for separation, then transferred onto polyvinylidene fluoride membranes. Membranes were blocked using 5% nonfat milk for ≥ 1 h, then incubated overnight at 4 °C with primary antibodies against ALOX12 (ab168384, abcam, 1:1000), COX2 (ab188184, abcam, 1:1000), p21 (ab109199, abcam, 1:1000), GPX4 (ab116703, abcam, 1:1000), FTMT (ab124889, abcam, 1:1000), HO-1 (ab13243, abcam, 1:1000), SLC7A11 (ab175186, abcam, 1:1000) and GAPDH (ab181602, abcam, 1:1000), and then rinsed three times with phosphate buffered saline (PBS) containing Tween (PBST), following by incubation with anti-rabbit or anti-mouse secondary antibodies for 1 h at 37 °C. Next, membranes were rinsed three times in PBST, and an Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA) used to visualize protein expression, with ImageJ analysis software applied to quantify grayscale values.