β actin

Ischemic cerebrum cells and tissues were lysed using a RIPA Lysis Buffer. Total protein was extracted using a Total Protein Extraction Kit (Takara) according to the manufacturer's instructions, and the quality was detected using the Bradford method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein extracts. An equal amount of protein was transferred onto a polyvinylidene fluoride membrane, and incubated with primary antibodies (anti-p-mTOR, anti-Beclin-1, anti-Mcl, 1:500, [Sigma, USA]) or β-actin (1:500, [R&D, China]) at 4°C for 24 h. The membranes were then incubated with the secondary antibody (1:1000) for another 2 h at room temperature. The ECL method and Image J software were used to visualize the bands. The antibodies were purchased from Abcam (UK). β-actin acted as an internal control.

Cells after SC99 treatment were prepared for immunoblotting according to our previous method [35 (link), 40 (link)]. A specific primary antibody against cyclin D₂ (CCND2) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antibodies against Bcl-2, Bcl-xL, Caspase-3, STAT3, p-STAT3(Tyr705), c-Src, p-Src, JAK2, p-JAK2, E2F-1, AKT, p-AKT, ERK, p-ERK, mTOR, p-mTOR, and PARP were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against VEGF, β-actin, α-tubulin, anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems (Minneapolis, MN). Anti-Myc antibody and GAPDH was purchased from Sigma (St. Louis, MO).

Western blot analysis was performed to detect the levels of CD126, gp130, ADAM10, ADAM17, and SOCS1–3 proteins, as well as total and phosphorylated JAK1, JAK2, TYK2, STAT1, and STAT3 in MDSCs and CD33⁺ control cells. Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for western blot analysis using mouse anti-human CD126, gp130 (R&D Systems, Inc., Minneapolis, MN, USA), SOCS1 (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), SOCS2–3 (R&D Systems, Inc.), and β-actin. Rabbit anti-human antibodies were used to detect JAK1, JAK2, TYK2, STAT1, STAT3, p-STAT1, p-JAK1, p-JAK2, p-TYK2, and p-STAT3 (Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated with primary antibodies overnight at 4°C, as described previously (7 (link)). Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG Ab (Zhongshanjinqiao, Beijing, China), and protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology, Rockford, IL, USA). The relative densities of protein bands were determined by comparing the band densities of proteins of interest to those of β-actin, using Quantity One software. We used the density ratio of phosphorylated protein to total protein to compare the expression of these phosphorylated proteins.

The antibodies to GPX4 and β-actin were obtained from R&D systems (Abingdon, UK) and Thermo Scientific (Dartford, UK) respectively. Curcumin, quercetin, rutin, EGCG, tannic acid and phytic acid were obtained from Sigma-Aldrich Company Ltd. (Dorset, UK) (Curcumin cat. no. C7727; EGCG cat. no. E3768). Erastin was purchased from Bertin Bioreagent (Montigny-le-Bretonneux, France).