Ab244205

We collected formalin-fixed paraffin-embedded sections from 149 patients who underwent surgical treatment and were confirmed to have ER+ BC at the Second Affiliated Hospital of Zhejiang University School of Medicine from January 2014 to June 2017. The inclusion criteria were defined as follows: (1) BC as a primary cancer diagnosis; (2) histological confirmation of BC; (3) curative operation performed; (4) ER positivity determined via immunohistochemistry (IHC) staining; and (5) complete clinicopathologic information. A 2-mm tissue core containing the dominant tumor area was collected for tissue microarrays. Collection of samples and clinicopathological information was undertaken after receiving informed consent and approval by the ethics committee. Staining scores were calculated by multiplying the proportion of positively stained tumor cells by the staining intensity. The samples were classified as having no (0), < 25% (1), 25–50% (2), 50–75% (3), or 75–100% (4) positive cells. The intensity was classified as no staining (0), weak staining (1), moderate staining (2), or strong staining (3). IHC staining was performed on 4-μm-thick sections, as previously described. The anti-ALOX15 monoclonal antibody used was purchased from Abcam (ab244205). Images were photographed by laser confocal microscopy.

Total protein was extracted from spleens of mice in each group using animal total protein extraction kit (Sangon Biotech, Shanghai, China, C510003). ~50 μg protein was loaded on 12% SDS-PAGE and transferred to nitrocellulose membrane (PALL, P/N66485, Waltham, USA). The membrane was sealed with 5% skim milk for 2 h and incubated with primary antibodies at 4 °C overnight. After washing the membrane with TBST buffer, the membrane was incubated with the secondary antibody at room temperature for 90 min. ECL developer solution was formulated (Thermo, VG299080) and imaging was performed using a gel image analysis imaging system (Fusion Solo, Vilber Lourmat, France). The antibodies for Hpgds (22522-1-AP) and Ptgs2 (66351-1-Ig) were purchased from Proteintech (Shanghai, China), while the antibody of Alox15 (Ab244205) was purchased from Abcam (Shanghai, China).

Ovarian tissues and GCs were lysed using the Tissue or Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China), and protein concentrations were detected using a BCA kit (Thermo, USA). Equal amounts of protein were dissolved in 5 × loading buffer, and electrophoresed on 7.5% or 10% SDS polyacrylamide gels and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with anti-FACL4 (1:10,000, ab155282, Abcam); anti-15Lipoxygenase antibody (1:1000, ab244205, Abcam); anti-xCT (1:1000, ab175186, Abcam); anti-GPX4 antibody (1:1000, TD6701, Abmart); anti-β-actin (1:20,000, Sigma-Aldrich) primary antibodies overnight. After incubation, the membranes were washed with TBS and Tween 20 (TBST) three times and then immunoblotted with HRP-conjugated secondary antibodies (1:2000, Proteintech) for 2 h at room temperature. The expression of each protein was measured by an enhanced chemiluminescence reagent (ECL) kit (Beyotime). The density of the protein expression bands was measured using ImageJ software.