Ab206414

According to the method described above, the washed platelets were obtained and resuspended in the HEPES-Tyrode buffer pre-warmed to 37 °C. The platelet suspension was successively incubated with 40 ng/mL of GDF-15 for 20 min at 37 °C and lysed with equal volumes of the 2 × NP-40 lysis buffer (300 mm NaCl, 100 mm Tris-HCl, 2% NP-40, 2 mm NaF, and 2 mm EDTA) containing protease and phosphatase inhibitors on ice for 30 min. The supernatant was collected after centrifugation at 12,000 rpm for 15 min, followed by incubation with the anti-GDF-15 antibody (ab206414, Abcam) or the isotype IgG control (ab172730, 7ed to each sample and incubated for 2 h at 4 °C. The precipitates were separated by centrifugation at 3000 rpm for 3 min and rinsed three times with the NP40 lysis buffer. Finally, the washed immunoprecipitated beads were boiled in loading buffers for further Western blot analysis. VeriBlot for the IP detection reagent (ab131366, 1:3000, Abcam), which only recognizes native (non-reduced) antibodies, was used for immunoblotting.

We combined the gene expression data of control samples (17 samples) with those of CRC samples (533samples) in the GSE39582 group and performed the Wilcoxon rank-sum test to further compare the differential expression of the 10 prognostic ferroptosis-related genes between the normal and tumor colon tissues. Besides, a total of 75-paired normal/tumor CRC specimens were recruited from Ruijin Hospital (Shanghai, China) following the guidelines set by the Ethical Committee of Ruijin Hospital. The tumor and adjacent normal colon tissues were fixed by 10% formalin and embedded by paraffin. The optimum sections of tissue specimens were selected and deparaffinized and immunohistochemistry (IHC) was implemented as the following antibodies: HAMP (Abcam, ab30760), FDFT1 (Abcam, ab195046), GDF15 (Abcam, ab206414), TFAP2C (Abcam, ab218107).

Equal amounts of whole-cell lysates were separated on a 10–12% SDS-polyacrylamide gel. The blotting membranes were probed using antiserum of GDF15 (Ab206414, Abcam, Cambridge, MA, USA), NDRG1 (42-6200; Invitrogen, Carlsbad, CA, USA), Maspin (#554292, BD Biosciences, San Jose, CA, USA), GFRAL (ab107719, Abcam, Cambridge, MA, USA), β-actin (MAB1501, Merck Millipore, Burlington, MA, USA), Smad 2/3 (SC-398844, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Smad 1 (#6944, Cell Signaling, Danvers, MS, USA), Smad 5 (#9517, Cell Signaling, Danvers, MS, USA), phospho-Smad 2/3 (#8828, Cell Signaling, Danvers, MS, USA), or phospho-Smad 1/5 (#9516, Cell Signaling, Danvers, MS, USA) antiserum. Band intensities were detected using the Western lightning plus-ECL detection system (PerkinElmer Inc., Waltham, MA, USA), recorded using the LuminoGraph II (Atto Corporation, Tokyo, Japan), and analyzed using the Image J [33 (link)].