Figure 2

H89 induces CARP-1 expression (A) and threonine phosphorylation (B), and depletion of CARP-1 abrogates apoptosis by H89 (C). In panel A, the cells were treated with noted dose of H89 or piceatannol for indicated time periods. Approximately 100 μg of respective protein lysates were electrophoresed on 10% SDS-PAGE, followed by western immunoblotting. The membranes were first probed with anti-CARP-1 (α2) antibody [3], followed by probing with anti-actin antibody to assess loading. In panel B, cells were either untreated (Control) or treated with H89 for indicated dose and time, and cell lysates were prepared. In lanes 2 and 3, co-immunoprecipitation using noted antibodies and 1000 μg protein lysate was carried out prior to western blotting. In lane 1, 100 μg protein lysate was analyzed along with immunoprecipitates on 10% SDS-PAGE followed by immunoblotting with anti-phospho-tyrosine or anti-phospho-threonine antibodies. In panel C, HBC cells expressing normal (indicated as CARP-1 antisense sublines 1, 4) or 50% reduced CARP-1 (indicated as sublines 9, 10) were either untreated or treated with noted time and dose of H89, and apoptosis determined as in figure 1D. Columns in histogram represent data from 3 independent experiments; bars, SE.