Stat5b

Cultured cells (THP-1-derived macrophages and RAW264.7) were lysed in NP-40 lysis buffer. This is composed of 1% NP-40 (Sigma), 20% Glycerol (Santa Cruz^®), 0.2 mM EDTA (Sigma), 40 mM HEPES (Gibco) pH 7.9, 0.5 M NaF (Sigma), 10 mM NaPpi (Sigma), 5 M NaCl (Fisher) and was supplemented with protease inhibitor cocktail (aprotinin, leupeptin, and pepstatin in a 1:1:1 ratio; Sigma), 0.5 M dithiothreitol (Sigma), 10 mg/mL phenylmethanesulfonyl fluoride (Sigma) and 100 mM Na₃VO₄ (Sigma). Debris was removed by centrifugation (Sorvall) at 1500 rpm for 15 min, and proteins were fractionated by SDS-PAGE, 8% polyacrylamide gel (BIO-RAD), and probed for RAGE, JNK, p-Akt, p-STAT3, STAT5b, and GAPDH (Santa Cruz^®). These were visualized with horseradish-peroxidase-coupled 2° antibodies—mouse-IgGκ, goat anti-rabbit IgG, or donkey anti-goat IgG (Santa Cruz^®) [62 (link),64 (link),65 (link)]—developed using western blotting substrates (Thermo Scientific^TM Pierce^TM ECL Plus Western Blotting Substrate, Rockford, IL, USA), and image-captured (FluorChem^TM 8900, ProteinSimple, San Jose, CA, USA). All experiments were done in triplicates. Densitometric analyses (ImageJ, version 1.53a [66 ]) were performed. Each band was quantified five times, and we reported the fold change of each exposure condition compared to media alone (MA), as described [63 (link)].

All chemicals, unless otherwise noted, were obtained from ThermoFisher or Merck. Enzymes were obtained from New England Biolabs. The following drugs/dyes were used for this work: IFNγ (final concentration of 50 ng/ml, Merck), dTAG13 (final concentration of 100 nM, Merck), Vybrant DyeCycle Ruby Stain (final concentration of 5 μM, ThermoFisher) and Tofacitinib citrate also known as CP‐690550 (JAK inhibitor, concentration of 10 μM, Merck). The following antibodies were used for this work: GBP5 (Cell Signaling Technology, 67798; Abcam, ab96119), STAT1 (Cell Signaling Technology, 9172), Phospho‐STAT1 (Ser727) (Cell Signaling Technology, 9177), Phospho‐STAT1 (Tyr701) (58D6) (Cell Signaling Technology, 9167), STAT2 (Santa Cruz Biotechnology, sc‐1668), STAT3 (Santa Cruz Biotechnology, sc‐8019), STAT5B (Santa Cruz Biotechnology, sc‐1656), IRF1 (Cell Signaling Technology, 8478), IRF9 (ThermoFisher Scientific, 702322), a‐TUB (Merck, T9026), anti‐rabbit (fluorophore‐conjugated) (LI‐COR, 926‐32211), anti‐mouse (fluorophore‐conjugated) (Rockland, 610‐744‐124), Rabbit IgG (Epicypher, 13‐0042k).

Phenol-red free RPMI 1640 media and RPMI 1640-GlutaMAX were purchased from ThermoFisher Scientific. Charcoal/Dextran treated Fetal Bovine Serum (FBS) was obtained from Atlanta Biologicals. STAT5a, STAT5b, STAT5, ERα, β-actin antibodies and AG-490 (JAK2 inhibitor) were obtained from Santa Cruz Biotechnology. Phospho (p)-ERK1/2, and U1026 (MEK1/2 inhibitor) were from Cell Signaling and the CDK7 inhibitor, THZ1 hydrochloride from Med Chem Express. pERα (Ser118) antibody was obtained from EMD Millipore. PRLR antibody were obtained from Sigma-Aldrich. Human PRL antibody were obtained from National Hormone and Peptide Program, Harbor-UCLA Med. Ctr., Torrance, CA 90502.

Cells were crosslinked, lysed and sonicated according to^{42 (link)}, then STAT5 And RNA polymerase II immunoprecipitation was performed according to^{43 (link)}. The antibodies used for immunoprecipitation were STAT5 A (sc-1081, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and STAT5 B (sc-835, Santa Cruz Biotechnology), 1.2 µg each, and RNA Polymerase II CTD (MABI0601, Clinisciences, Nanterre, France), 2 µg. DNA from ChIP was isolated with a phenol/chloroform extraction, and used for qPCR with the primers listed in Supplementary Table 1. Results were then normalized using the percent input method.