Anti gata3 sc 269

To evaluate the expression levels of GATA3 and AGR3 in tumor tissues of grade III ER⁺HER2⁻ patients, IHC was performed using anti‐GATA3 (sc‐269; Santa Cruz) and anti‐AGR3 (sc‐390940; Santa Cruz). Tissue sections were fixed with 4% formaldehyde and embedded with paraffin. The expression levels (i.e., positive or negative) were assessed by mean density using image pro plus software.

After fixation in 1 % formaldehyde, T cells (1 × 10⁷) were lysed for 5 min in 50 mM Tris buffer (pH 8) containing 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.1 % Nonidet P (NP)-40 and 10 % glycerol supplemented with anti-protease cocktail (Roche Life Science, Indianapolis, IN, USA). Nuclei were resuspended in 50 mM Tris buffer with 1 % SDS and 5 mM EDTA. Chromatin was sheared by sonication. After preclearing with protein A beads (Santa Cruz Biotechnology), lysates were incubated overnight at 4 °C with 1 μg/ml anti-GATA-3 (sc-269) or mouse immunoglobulin G (IgG) antibodies (Santa Cruz Biotechnology). Immune complexes were collected with protein A, washed three times with high-salt buffer (50 mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % sodium deoxycholate), twice with low-salt buffer (10 mM Tris–HCl, 250 mM LiCl, 1 mM EDTA, 0.5 % NP-40, 0.1 % sodium deoxycholate) and then twice with Tris-EDTA (TE). Immune complexes were extracted in 1× TE buffer, and protein crosslinking was reverted by heating at 65 °C for 5 h. DNA was then extracted by phenol-chloroform and precipitated by ethanol, and a 1/20 fraction of the immunoprecipitated DNA was used for qRT-PCR.