Preparation and Analysis of Cell Lysates

Preparation of lysates and immunoblot analyses were performed as described previously (5 (link)) using Tris lysis buffer (50 mM Tris–HCl pH 7.8, 150 mM NaCl, 1% IGEPAL CA-630) containing 20 mM NaF, 20 mM β-glycerophosphate, 0.3 mM Na-vanadate, 20 μg/ml RNase A, 20 μg/ml DNase and 1/300 protease inhibitor cocktail (P8340, Sigma–Aldrich) and phosphatase inhibitor cocktail #2 (P5726, Sigma–Aldrich). Antibodies used in this study were purchased from the following sources: rabbit anti-Cdk2 (M2 SC-163, Santa Cruz Biotechnology), mouse anti-actin (ab-3280, Abcam, Cambridge, MA, USA), rabbit anti-GFP (ab-290, Abcam), mouse anti-tubulin (Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-Cyclin A (SC-751, Santa Cruz Biotechnology) and mouse anti-cyclin E (SC-198, Santa Cruz Biotechnology). Secondary antibodies used for western blot analysis were goat anti-mouse (31430) and, goat anti-rabbit (31460, Thermo Scientific). mouse anti-tubulin hybridoma cell line (clone #12G10) was developed by J. Frankel and E.M. Nelson under the auspices of the NICHD and maintained by the Developmental Studies Hybridoma Bank.