Gst antibody

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.

Peptide arrays were synthesized as previously described (Fuchs and Strahl, 2011 (link); Rothbart et al., 2012 (link)). Bromodomains were cloned into pGEX-6P1 vectors, expressed in BL21 E. coli, and purified using batch purification with glutathione resin (Pierce). Proteins were eluted and dialyzed in PBS. Domains were arrayed as previously described using GST antibody (Sigma) to detect the bound protein (Rothbart et al., 2012 (link)). H4 acetyl antibody (Millipore 06–866, Lot 2491213) specificity was determined as previously described (Rothbart et al., 2015 (link)) using 1:2000 dilution in PBS with 0.1% Tween-20 and incubation for 1 h at room temperature. After incubation with domains or antibody, arrays were washed in cold PBS, incubated in secondary antibody and scanned (Typhoon, GE). After normalizing the average signal of triplicate spots to the peptide with the highest signal, heatmaps were generated with Java TreeView with a range of intensity from 0 to 1 (Saldanha, 2004 (link)).