Lambda phosphatase

IP products from mULK1-3FLAG-overexpressing HEK-293T cells were aliquoted and treated with reaction buffer, reaction buffer containing 1 μL (400 units) lambda phosphatase (P0753S, NEB), or reaction buffer containing 1 μL lambda phosphatase plus 1×phosphatase inhibitors cocktail (Roche) at 30 °C for 30 minutes with gentle shaking. Then the reaction products were denatured at 95 °C for 7 minutes and subjected to immunoblotting with FLAG or Serine/Threonine-specific antibody.

Brains were dissected from the heads of 5 worker pupae under binocular microscopy and homogenized in 150 µl buffered insect saline [20 mM Tris-HCl (pH 7.4), 130 mM NaCl, 5 mM KCl, 1 mM CaCl₂, containing protease inhibitor cocktail (Roche)]. 2 ul (800 Unit) of Lambda Phosphatase (Santa Cruz Biotechnology, USA), 10 µl of 10x Lambda Phosphatase buffer and 10 µl of 10x MnCl₂ solution (Santa Cruz Biotechnology) were added to the 65 µl homogenate and total volume was made up to 100 µl with water. The sample was incubated at room temperature for 30 min. As a negative control, in which dephosphorylation was inhibited, 10 µl of 10x PhosSTOP (Roche) was added instead of Lambda Phosphatase. After the incubation, the samples were used for immunoblotting.