Pc jun

For reverse western blot, Proteome Profiler Antibody Array Kits for human angiogenesis factors, chemokines, cytokines and phospho-kinases (R&D systems) were used, according to the manufacturer’s instructions. For western blot, 400,000 MDA-MB-231 or LECs (per well) were starved for 24 h, after which they were treated with Stattic (5–10 µM), S3I-201 (2.5–10 µM), or SP600125 (40 µM) and incubated for 60 min. After that, inducers, including TCM (30%), EGM, IL6-dep-TCM, IL6 or EGF were added. We followed the standard protocol for the rest of the procedure as described previously^{11 (link)} applying antibodies of interest, including pStat3, HIF-1α, gp130, pNFkB, NFkB, IkBα, Stat3, pCREB, GAPDH (all from Cell Signaling), pc-Jun, pATF-2 (Sigma), CCR5, and Lamin B1 (Abcam). All the original gel images of immunoblot analyses are presented in Supplementary Fig. 25.

LECs (2×10⁶) treated with Stattic, SP600125, IL6 or EGM, were used to prepare cell lysates or nuclear extracts. Five hundred microliters of cell lysates or 200 µL nuclear extracts were incubated overnight at 4°C with antibodies suitable for IP (1:100 diluted): pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Cell Signaling). Ten microliters of Protein A/G Plus Agarose (Santa Cruz Biotech) was added and incubated for 3 h at 4°C. The beads were rinsed 3 times with 500 µL cell lysis buffer for IP (Pierce) supplemented with the protease inhibitor and phosphatase inhibitor cocktail 2/3 (Sigma). The protein complex was reduced and separated by SDS-PAGE and probed with the following antibodies in a Western assay: pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Sigma). All the original gel images of immunoblot analyses are presented in Supplementary Fig. 25.