C fos h 125

MDA-MB 231 and SUM159 cells were grown in 10 cm dishes, exposed to the treatments and then lysed as previously described [48 (link)]. Equal amounts of whole protein extract were electrophoresed through a reducing SDS/8 and 10% (w/n) polyacrylamide gels, electroblotted onto a nitrocellulose membrane (Amersham Biosciences, GE Healthcare, Milan, Italy), and probed with primary antibodies against Y397-FAK (Cell Signaling Technology, Milan, Italy), FAK (H-1) (Santa Cruz Biotechnology, DBA, Milan, Italy), phosphorylated ERK1/2 (E-4) (Santa Cruz Biotechnology, DBA, Milan, Italy), ERK2 (C-14) (Santa Cruz Biotechnology, DBA, Milan, Italy), p-AKT1/2/3 (Ser 473)-R (Santa Cruz Biotechnology, DBA, Milan, Italy), AKT/1/2/3 (H-136) (Santa Cruz Biotechnology, DBA, Milan, Italy), c-FOS (H-125) (Santa Cruz Biotechnology, DBA, Milan, Italy), EGR1 (C19) (Santa Cruz Biotechnology, DBA, Milan, Italy), CTGF (Origene, DBA, Milan, Italy) and β-actin (C2) (Santa Cruz Biotechnology, DBA, Milan, Italy). Proteins were detected by horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology, DBA, Milan, Italy) and then revealed using the ECL™ Western Blotting Analysis System (GE Healthcare, Milan, Italy).

The antibodies used were as follows: NF-κB p65 (Abcam; cat. no. ab7970), Pol II (Santa Cruz; cat. no. SC-899 and SC-900, mixed in a 1:4 ratio), β-tubulin (Abcam; cat. no.ab6046), SNRP70 (Abcam; cat. no ab51266), c-Fos (H-125, Santa Cruz; cat. no. sc-7202), c-Jun (H-79, Santa Cruz; cat. no. sc-1694), and NF-κB p50 (Abcam; cat. no. ab7971).

SkBr3 cells, CAFs and tumor homogenates obtained from nude mice were processed according to the previously described protocol.^{67 (link), 68 (link), 69} Protein lysates were electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane probed with primary antibodies against SIRT1 (D739) and acetyl-p53 (Lys382) purchased from Cell Signaling Technology, Euroclone (Milan, Italy), c-fos (H-125), phosphorylated ERK1/2 (E-4), ERK2 (C-14), EGFR (1005), p-EGFR^Tyr1173 (sc-12351), p21 (H164), GPER (N-15), Cyclin D1 (M-20), Ki-67 (H-300) and β-actin (C2) purchased from Santa Cruz Biotechnology (DBA, Milan, Italy). The levels of proteins and phosphoproteins were detected, after incubation with the horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology), by the ECL System (GE Healthcare, Milan, Italy).