Fig. 7.
Cmpd-3 increases NE-mediated phosphorylation of CREB in the presence of α2-and β-AR blockers. (A) Western blot analysis of pSer133-CREB levels in rat-1 fibroblasts transfected with the α1A-AR. Cells were preincubated for 2 h with β-AR inhibitor, propranolol (10 μM) and the α2-AR inhibitor rauwolscine (0.1 μM), with or without the addition of Cmpd-3 (10−8 M) in the presence of 500 μM IBMX. Cells were stimulated by NE (10−4 M) for an additional 2 h before subjected to SDS-PAGE. The transferred blot was probed with rabbit p-CREB antibody (pSer133, Cell Signaling #9198) at 1:1000, then 1:10,000 goat anti-rabbit IgG HRP in 5% BSA in PBST. The blot was stripped and incubated with rabbit GAPH antibody (Cell Signaling # 2118) as a loading control. (B) The bands were quantified using the Image Studio Digits Version 4.0 program associated with the Li-Cor digital scanner model C-DIGit, normalized to GAPDH, and graphed using Prism software. P-CREB is identified at 43 kDa, and a phosphorylated form of the CREB-related protein ATF-1 also cross reacts (29 kDa). pRepresentative gel. N = 12, from 3 independent experiments performed in quadruplet. ∗P < 0.008.