Rabbit anti β actin

Xenopus embryos were homogenized in lysis buffer [20 mM Tris–HCl: pH 8.0, 5 mM MgCl₂, 1 mM EDTA, 50 mM KCl, 10% glycerol, 0.5% Triton-X, Protease Inhibitor Cocktail (Roche Applied Science)], and embryonic protein extracts were used for immunoblotting with rabbit anti-γ-tubulin (1:1000, abcam), mouse anti-centrin (1:1000, Millipore) and rabbit anti-β-actin (1:2000, Thermo Scientific) antibodies.

MOVAS-1 cell lysates from 1 h, 12 h, and 24 h old cultures (30 µg of protein) were separated on a 12% polyacrylamide gel under reducing conditions and transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham, Mississauga, Ontario, Canada). The membrane was blocked with 5% non-fat milk in PBS and probed with either rabbit anti-phospho-ERK1/2 (1:1000, Abcam), rabbit anti-ERK1/2 (1:1000, Abcam), or rabbit anti-β actin (1:5000, ThermoFisher, Burlington, Ontario, Canada) antibodies in PBS, 5% non-fat milk, and 0.1% Tween 20 overnight at 4 °C. Anti-rabbit IgG-HRP (Abcam) was used as a secondary antibody and membranes were incubated for 1 h at room temperature. Finally, membranes were developed by chemiluminescence (Amersham).

Proteins from uteri were extracted at the indicated time points in RIPA buffer using homogenizer, and suspended in Laemmli loading buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% β-mercaptoethanol). The proteins were then separated on a 12% acrylamide gel, followed by their transfer to a nitrocellulose membrane. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies over-night at 4 °C (rabbit anti-FOXL2 1:1000, Bioss, Woburn, MA; rabbit anti-β-ACTIN 1:2000, Thermo Scientific, Waltham, MS), then with the secondary antibodies for 1 h at room temperature (anti-rabbit 1:5000, Jackson laboratory, Bar Harbor, MI). The immunoreactive bands were detected by ECL (Amersham, England).