α foxl2

COS-7 or COV434 cells were lysed 48 h after transfection in 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (pH 7.6) supplemented with protease inhibitors PMSF 1 mM and Complete mini EDTA-free cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitors (Sigma Aldrich, St Louis, MO, USA). Clarified lysates were used for Western blotting or immunoprecipitated using Anti-V5 Agarose Affinity Gel (Sigma Aldrich, St Louis, MO, USA) according to instructions. Precipitated proteins were eluted in 2x-SDS sample buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol and 0.1% bromophenol blue). 100 mM DTT was further added to the samples. Eluates were separated by SDS-PAGE using NuPage Bis-Tris 4–12% Gels (Invitrogen Corporation) and proteins were electrotransfered onto PVDF membranes (Hybond-P, GE Healthcare, Waukesha, WI, USA). Antibodies used for detection were: α-GATA4 1∶1000 (sc-1237, Santa Cruz Biotechnology,), α-FOXL2 1∶1000 (IMG-3228, Imgenex) and α-SMAD3 1∶1000 (#51–1500, Invitrogen Corporation).

GCT tissue microarray and normal ovarian samples were stained as previously described and presented for GATA4 and CCND2 [25] (link), [26] (link), with following primary antibodies: α-GATA4 1∶400 (sc-1237, Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-FOXL2 1∶400 (IMG-3228, Imgenex, San Diego, CA, USA), α-SMAD3 1∶400 (#51–1500, Invitrogen Corporation, Carlsbad, CA, USA), and α-CCND2 1∶1000 (sc-593; Santa Cruz Biotechnology). The GCT stainings were classified into three groups based on the intensity of staining: high representing intermediary or strong intensity in 70% of the cells, intermediate representing moderate or strong intensity in <70% of the cells, and low representing negative or low intensity even if detected in 100% of the cells; scoring of GATA4 staining as described (34), i.e. high for 80–100% positive nuclei, intermediate for 20–80% positive nuclei, and low for 0–20% positive nuclei. Two researchers (M.A. and N.A. or A.F) independently performed the evaluation and disagreements were resolved by a joint review.