Phospho rsk2

The nonprostanoid EP4 receptor agonist CP-734432 was obtained from Pfizer Global Research and Development (Groton, CT). For in vitro studies, it was used at a final concentration of 10⁻⁶ M. Cells were lysed with golden lysis buffer (20 mM Tris, 137 mM NaCl, 5 mM Na₂EDTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, 1 mM leupeptin, 1 μM pepstatin A, 10 mM NaF, 1 mM Na₃VO₄, 1 mM EGTA, 1 mM tetrasodium PP1, and 100 μM P-glycerophosphate). Western blot was performed with the invitrogen system. Chemiluminescence was done with West Femto reagent (Thermo Fisher Scientific, Rockford, IL). The following antibodies were used in the experiments: COX-2 (Cayman Chemicals), ATF4 and phospho ATF4 (Abcam, Cambridge, MA), phospho ERK1/2 and phospho RSK-2 (Cell Signaling Technology, Boston, MA). The experiments were usually repeated for three times. After establishing the trends of protein expression, the clear results with less variation were chosen and represented. Immunoprecipitation (IP) was performed using the Catch and Release Reversible IP System (EMD Millipore). The protein was pulled down with V5 antibody (Invitrogen) and GST antibody (Sigma) was used as antibody control. IP was performed using antibodies against phospho-EKR1/2 and RSK-2.