Ripa buffer

To identify the endogenous phosphorylation sites in mouse brain DGK-θ, 1–2 ml of RIPA buffer (pH 7.6, Thermo Scientific) was added to each mouse brain and homogenized using a 7 ml mortar and pestle until it appeared homogenous. It was then incubated in the RIPA buffer at 4°C for 15 min followed by 15s sonication using a Branson Digital Sonifier SFX250 equipped with half half-inch horn. The lysate was then centrifuged at 17000g for 10 min, and the enzyme was immunoprecipitated from the supernatant using an anti-DGK-θ antibody purchased from Santa Cruz. After five washes using RIPA buffer, DGK-θ along with other proteins on the beads were eluted off by boiling sample buffer at 85°C for 5 min. The immunoprecipitated sample was subjected to 8% SDS-PAGE, and the DGK-θ band was identified by Western blot analysis of a sister blot. The DGK-θ band was cut from the gel, reduced, alkylated, and trypsin digested as described below.

Proteins were extracted with RIPA buffer (Sigma-Aldrich) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Briefly, cells were trypsinized from culture dishes, centrifuged, and washed once with cold PBS. Cell pellets were then resuspended and incubated in RIPA buffer for 20 min on ice and sonicated with 3 pulses 10 sec each at 10% amplitude (Branson Digital Sonifier, Danbury, CT, USA). After centrifugation, supernatants were denatured in Laemmli loading buffer for 10 minutes at 98°C. Total protein (20 μg) was separated by SDS-PAGE and transferred to PVDF membranes which were subsequently probed with anti-Nanog (Bethyl, Montgomery TX, USA), anti-Oct4 (BD Transduction, San José, CA, USA), anti-Pdx1 (Abcam, Cambridge, UK), anti-Sox17 (Millipore, Billerica, MA, USA), or anti-Gata4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (Sigma-Aldrich) as loading control.