Sc 2056

Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, 0.5% Na-Deoxycholate, 0.1% SDS, and 1% NP-40), and an equal amount of protein was resolved using Nupage Novex 4–12% Bis-Tris Gel (Thermo Fisher Scientific) as previously described in ref. ^{22 (link)}. Primary antibodies used were β-ACTIN (ACTB) (1:5,000, #2228; Sigma), ATRX (1:1,000, sc-15408; Santa Cruz Biotechnology), Flag (1:1,000, F1804; Sigma), H3 (1:2,000, ab1791; Abcam), H3K36me2 (1:1,000, 2901; Cell Signaling), H3K36me3 (1:2,000, 61101; Active Motif), KDM2A (1:1,000, A301-476A; Bethyl Laboratories), SENP6 (1:500, HPA024376; Sigma-Aldrich), SMC5 (1:2000, A300-236A; Bethyl Laboratories), and TUBULIN (1:2,000, ab15246; Abcam). Secondary antibodies used were donkey anti-rabbit HRP (1:1,000, sc-2077; Santa Cruz Biotechnology), donkey anti-mouse HRP (1:1,000; sc-2096, Santa Cruz Biotechnology), and donkey anti-goat HRP (1:1,000, sc-2056, Santa Cruz Biotechnology). Antibody signal was detected using the ECL Western Blotting Substrate (W1015, Promega) and X-ray film (F-BX810, Phenix).

Uterine tissue was dehydrated through a graded ethanol series, cleared in toluene, and then embedded in paraffin. We cut 7-μm cross sections on a microtome and mounted on Shandon polysine precleaned microscope slides (6776215Cs, Thermo Fisher). Sections were stored in the dark at room temperature until they were stained. We localized the expression of FOXO1 using immunohistochemistry. Slides were deparafinized in three successive washes of xylene (3 min each), then three successive washes of ethanol (3 min). Antigen retrieval was performed in citrate buffer (12 mM sodium citrate, pH 6.0, 98°C, 1 h). Endogenous peroxidase activity was blocked with Dako Peroxidase Block (Dako, 30 min). Slides were then incubated in primary antibody overnight. We used a goat polyclonal antibody generated against the N-terminal of human FOXO1 protein (1:5,000 dilution, FKHR antibody sc-9809, SantaCruz). On day two, slides were incubated in a donkey anti-goat IgG-HRP secondary (1 h, 1:200 dilution, sc-2056 SantaCruz). Slides were then rinsed in PBS (5 min), PBS-BSA (0.1%, 5 min), then incubated for 5 minutes in TSA Plus Cyanine 3 system (1:50 NEL744001KT, PerkinElmer Inc.). Slides were again washed in PBS (5 min) and PBS-BSA (0.1%, 5 min), counter stained in DAPI (1 time, 2 min, 10236276001; Roche), washed in deionized water for 5 min, and mounted in glycerol (50%).