CREB is required for inducing p190-A promoter activity. (A) Thrombin activates CREB. HPAE cells were stimulated with thrombin (50nM) for the indicated times, and phosphorylation of CREB was determined by immunoblotting using Ser133-phosphospecific antibody. Membrane was reprobed with anti-CREB antibody to normalize for protein loading. Data represent mean ± SD from 3 individual experiments. Asterisk (*) indicates significant increase in phosphorylation above time 0 (P < .05). (B) Thrombin induces CREB nuclear localization. HPAE cells stimulated with thrombin for indicated times were lysed, and nuclear extracts were prepared as described in “Electrophoretic mobility shift assay.” Nuclear extracts containing equal amount of proteins were analyzed for protein-CRE DNA-binding activity using 32P-labeled consensus CRE oligonucleotide as a probe. (C) Effect of CREB knockdown on p190-A promoter expression. Nuclear extracts from cells transducing control vector or dn-CREB mutant were used for ChIP assay using monoclonal anti-CREB antibodies, and the resulting DNA fragments were subjected to PCR amplification using primers spanning the CREB consensus sequences from human p190-A. (D) Depletion of CREB suppresses p190-A promoter activity. HPAE cells expressing Sc or SiCREB for 24 hours were transfected with p190-A luciferase promoter construct. Cells were then left unstimulated or stimulated with thrombin for 6 hours, and the luciferase activities were determined. Asterisk (*) indicates values different from values from SiSc group at time 0 (P < .05), and double asterisk (**) indicates value different from SiSc cells after without or with thrombin stimulation.
