Anti vdr receptor

Ovarian tissues were immediately washed with ice 0.9% saline solution (w/v), weighed and homogenized in a volume of 100 mg tissue/300 μL of lysis buffer (0.1 M Tris, 0.01 M NaCl, 0.025 M EDTA, 1% NP40, 1% Triton X100, Sigma-Aldrich, Milan) supplemented with 2 mM sodium orthovanadate, 0.1 M sodium fluoride (Sigma-Aldrich, Milan), 1:100 mix of protease inhibitors (Sigma-Aldrich, Milan), 1:1000 phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, Milan), using an electric potter at 1600 rpm for 2 min. Samples were mixed for 30 min at 4 °C, centrifuged for 30 min at 13000 rpm at 4 °C and 40 μg of proteins for each samples resolved on SDS-PAGE gel at 15%. Proteins transferred to polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy) were incubated overnight at 4 °C with specific primary antibody: anti-VDR receptor (1:400, Santa-Cruz), anti-ERβ (1:500, Santa-Cruz), anti-cyclin-D1 (1:1000, Euroclone, Milan, Italy). Protein expression was normalized and verified through β-actin detection (1:5000; Sigma-Aldrich) and expressed as a mean ± SD (%).

CHO-K1 cells were lysed in ice Complete Tablet Buffer (Roche) supplemented with 2 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich), 1:50 mix Phosphatase Inhibitor Cocktail (Sigma-Aldrich) and 1:200 mix Protease Inhibitor Cocktail (Calbiochem). 35 μg of proteins of each sample were resolved on 10% SDS-PAGE gel. Polyvinylidene difluoride membranes (PVDF, GE, Healthcare Europe GmbH, Milan, Italy) were incubated overnight at 4 °C with specific primary antibody: anti-VDR receptor (1:400, Santa-Cruz) and anti-ERβ (1:500, Santa-Cruz). Protein expression was normalized to the specific total protein (if possible) and verified through β-actin detection (1:5000; Sigma-Aldrich) and expressed as a mean ± SD (%).