Ab252833

The injured spinal cord was harvested and digested, and the tissue lysate protein concentration was determined using a bicinchoninic acid (BCA) protein quantitation kit (Thermo Scientific). After conventional electrophoretic separation, membrane transfer, and blocking, primary antibodies against Glutathione Peroxidase 4 (GPX4) (ab252833, abcam), HO-1 (ab305290, abcam), Nrf2 (ab92946, abcam) and secondary antibodies were sequentially applied. Pierce ECL Plus Western Blotting Substrate was indicated to assess the fluorescence intensity, and ImageJ software was used for quantitative identification.

Protein samples from RAW264.7 macrophages or lung tissues were washed with PBS, followed by the lysis on ice in lysis buffer containing phosphatase inhibitors and protease inhibitors. Next, the samples were centrifuged (12,000 × g) at 4°C for 15 min. Equal amounts of protein were separated on SDS-PAGE gel, which were then transferred onto polyvinylidene difluoride membranes. After that, 5% nonfat milk was used to block the membranes for 90 min at room temperature, followed by the incubation with primary antibodies at 4°C overnight. Then, the membranes were incubated with peroxidase-conjugated secondary antibodies at room temperature for 60 min. At last, the grey value of these protein bands was scanned and quantified using Image Lab software (BioRad, USA). The primary antibodies as well as their dilution ratio are displayed as follows: SOD1 (#ab183881, Abcam, 1 : 1000), SOD2 (#ab68155, Abcam, 1 : 1000), GPX4 (#ab252833, Abcam, 1 : 500), GAPDH (#ab8245, Abcam, 1 : 1000), phosphorylated-AKT (p-AKT) (#ab38449, Abcam, 1 : 500), AKT (#ab18785, Abcam, 1 : 500), p-Foxo1 (#ab259337, Abcam, 1 : 500), and Foxo1(#ab179450, Abcam, 1 : 500). The relative protein levels of genes were normalized with the level of GAPDH in the same group.

The ischemic penumbra of rat brain tissues (n = 6) was collected for Western blot analysis as previously described (Liu et al., 2020 (link)). The following primary antibodies were used: anti‐beta‐Actin (Abcam, ab8226, 1:1000), anti‐GPX4 (Abcam, ab252833, 1:1000), anti‐FTH1 (Abcam, ab183781, 1:1000), anti‐TF (Abcam, ab82411, 1:1000), anti‐TF receptor (Abcam, ab269513, 1:1000), anti‐HO1 (Abcam, ab68477, 1:1000), anti‐ACSL‐4 (Abcam, ab155282, 1:1000), and anti‐xCT (Invitrogen, PA1‐16893, 1:1000).

The total proteins were extracted from heart tissues, or cells by using ice-cold RIPA lysis buffer (Beyotime, China). A total of 30 μg proteins were separated by SDS-polyacrylamide gel (SDS-PAGE), shifted to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany), and blocked in 5% non-fat milk, followed by incubation with specific primary anti-collagen 1 (ab254113, Abcam, United States, 1:1,000), anti-α-SMA (ab40854, Abcam, United States, 1:1,000), anti-GPX4 (ab252833, Abcam, United States, 1:1,000), anti-SLC7A11 (ab175186, Abcam, United States, 1:1,000) antibodies at 4°C overnight. Next day, the protein bands were incubated with HRP conjugated secondary anti-mouse or anti-rabbit antibodies (ab175775/ab288151, Abcam, United States, 1:1,000) at room temperature for 1 h. The bands were then visualized by using ECL (Thermo) on a gel image system (Bio-Rad, United States). All antibodies were purchased from Abcam.