Figure 2.

PKC inhibition prevents the cytoplasmic accumulation of HuR by ATP. (A) Representative Western blot analysis showing stimulus-dependent changes in the cytoplasmic (top) and total HuR levels (bottom) in hMC. Cells were serum-starved for 16 h and subsequently remained untreated (−) or were treated for additional 4 h with 30 μM ATPγS in the absence (vehicle) or presence of either 20 μM UO126, 10 μM SB203580, 30 μM PD98059, or 100 nM staurosporine as indicated. All inhibitors were preincubated for 30 min before the addition of ATPγS. Protein lysates (50 μg) from cytoplasmic (top) fractions or whole cell extracts (bottom) were subjected to SDS-PAGE and immunoblotted with an anti-HuR–specific antibody. To correct for variations in the protein loading, the blots were stripped and reincubated with an anti-β-actin antibody. (B) Effects of different PKC inhibitors (top) or PKC activators (bottom) on ATP-induced HuR accumulation in cytoplasmic fractions. hMC were serum starved for 16 h and subsequently remained untreated (−), or they were treated for additional 4 h with 30 μM ATPγS in the absence (+vehicle) or presence of either 20 nM Gö6976, 100 nM CGP 41251, or 10 μM rottlerin. hMC were treated for 4 h with either vehicle, 30 μM ATPγS, 50 nM bryostatin, or 100 nM PMA as indicated, and HuR cytoplasmic levels were assessed by Western blot analysis. The Western blots shown are representative of two independent experiments giving similar results.