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. 2012 Nov 26;32(41):4892–4902. doi: 10.1038/onc.2012.517

Figure 2.

Figure 2

Notch activates JAK/STAT signaling via upregulation of IL-6. (a) Addition of recombinant IL-6 to the cell culture medium of MDA-MB-231 cells (+ rec IL-6) led to elevated levels of pTyr705 STAT3 (phosphorylated STAT3 (pSTAT3)) but not total STAT3 (tSTAT3) protein. (b) Analysis of pSTAT3 (upper) and total STAT3 (lower) protein levels in MDA-MB-231 cells infected with adenoviral vectors expressing Notch1 ICD or enhanced green fluorescent protein (EGFP) (control) as indicated. In the two rightmost lanes, an IL-6 blocking antibody (α-IL-6) was added to the cell culture medium before Notch1 ICD or EGFP expression. (c, d) Analysis of (c) Bcl-xL protein levels in MDA-MB-231 cells infected with adenoviral vectors expressing Notch1 ICD or EGFP (control) or (d) in MDA-MB-231 cells supplemented with recombinant IL-6, and with phosphate-buffered saline (PBS) as control. (e) Analysis of paracrine IL-6 activation in MCF7 cells. Conditioned medium from MDA-MB-231 cells transfected with Notch1 ICD or EGFP (control). The conditioned medium was pre-treated with an IL-6 blocking antibody (α-IL-6) before supplemented to the MCF7 cells, and the levels of pSTAT3 and total STAT3 protein were analyzed.