Lambda protein phosphatase

Stably transfected cells were plated onto poly-L-lysine-coated 60-mm dishes and grown for 2 days to 80% confluency. After treatment with agonists or antagonists, cells were lysed with detergent buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 10 mM NaF; 10 mM disodium pyrophosphate; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS) in the presence of protease and phosphatase inhibitors. Where indicated, cells were preincubated with GRK2/3 inhibitor compound 101 or NOP receptor antagonists for 30 min before agonist exposure. After 30 min centrifugation at 4 °C, HA-tagged hNOP receptors were enriched using anti-HA-agarose beads. Samples were inverted for 1.5 hours at 4 °C. Where indicated, samples were dephosphorylated with lambda protein phosphatase (Santa Cruz) for 1 hour at 30 °C. After washing, proteins were eluted using SDS sample buffer for 30 min at 50 °C. Proteins were separated on 7.5% or 12% SDS-polyacrylamide gels, and after electroblotting, membranes were incubated with 0.1 μg/ml antibodies to pSer³⁴⁶ (5034), pSer³⁵¹ (4876) or pThr³⁶²/Ser³⁶³ (4874) overnight at 4 °C, followed by detection using enhanced chemiluminescence detection (ECL) of bound antibodies (Thermo Fisher Scientific). Blots were subsequently stripped and reprobed with the phosphorylation-independent antibody to NOPR (4871) or antibody to HA-tag (0631) to ensure equal loading of the gels.