Western blot analysis
Embryos (E13.5) for protein isolation were homogenized in lysis buffer containing 1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (CompleteMini; Roche). Equal amounts (25 μg) of protein lysate were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore, Billerica, USA) according to standard protocols. Western blots were done using a specific antibody to Ptdsr (PSR N-20, sc-11632; Santa Cruz Biotechnology Inc., Santa Cruz, USA) and β-actin (ab-6276; Abcam, Cambridge, UK) as described by the supplier. Secondary antibodies conjugated to horseradish peroxidase were from Santa Cruz and Abcam, used as described by the supplier, and detection was performed with an enhanced chemiluminescence system (ECLPlus; Amersham).