Anti gr antibody m20 sc 23476

BZ cells cultured in x-well tissue culture chambers (Sarstedt, Numbrecht, Germany) were fixed with PBS containing 4% paraformaldehyde (Sigma-Aldrich) solution for 15 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 5 min and blocked by 1% non-fat milk in PBS with 0.1% Tween-20 (PBST) for 1 h at 37 °C. Cells were incubated with anti β-tubulin III antibody TU20 (sc-51670, Santa Cruz, La Jolla, CA, [49 (link)]) or with anti-GR antibody M20 (sc-23476, Santa Cruz, [50 (link)]) overnight at 4 °C and then with secondary antibody Cy3 (Interchim, Thermo scientific) for 45 min in PBST with 1% milk. After washing, BZ cells were incubated with DAPI (4′,6′-diamidino-2-phenylindole), 1: 1000 in PBST for 2 min and observed with a fluorescence Olympus microscope AX70 (Olympus, Hambourg, Germany).

Immunoprecipitations were performed using 30 μl protein A-coated magnetic beads (Diagenode). Beads were washed 3 times with C1 buffer of the High Cell ChIP kit (Diagenode) and incubated for 4 h with anti-GR antibody H300 (Santa Cruz) at 4 °C with protease inhibitors and 0.2% BSA. Then, 350 μg of proteins from BZ cell lysates were incubated overnight with antibody-coated beads. The following day, beads were washed 3 times with buffer C1 and once with buffer W1 (High Cell ChIP kit), and immunoprecipitated proteins were eluted from the beads with 20 μl of Laemmli buffer at 95 °C for 5 min and loaded on a 10% SDS-PAGE gel for Western blotting. Membranes were hybridized with anti-GR antibody M20 (sc-23476, Santa Cruz, [54 (link)].