FIGURE 8.
Effect of nucleotides on the complex formation of histatin 3-HSC70-p27Kip1. A, association of HSC70 with p27Kip1 in the presence or absence of histatin 3 and nucleotides. HGFs were cultured in DMEM containing 0.1% FBS over 24 h. Proteins extracted from cells were immunoprecipitated with anti-p27Kip1 antibody, and precipitates were treated with P3a and histatin 3 followed by incubation with ATP, ADP, and ATPγS. Western blotting (WB) was then carried out using anti-HSC70 (top) and anti-p27Kip1 (bottom) antibodies. B, effect of nucleotides on the association of HSC70-p27Kip1 with histatin 3. HEK293 cells were transfected with the expression vectors encoding T7-tagged HSC70 and T7-tagged HSC70(D10N). Proteins in cell extracts were mixed and precipitated with GST-histatin 3. Precipitates were incubated with ATP, ADP, and ATPγS, and Western blotting was carried out using anti-T7 (top), anti-p27Kip1 (middle), and anti-GST (bottom) antibodies.