Figure 2.

HSP90, CHIP, and p14ARF form a ternary complex. A, Plasmids expressing p14ARF-FLAG, Myc-CHIP, and HA-HSP90β were transfected into 293T cells as indicated. After 24 hours, immunoprecipitation assay was performed using cell lysates. HSP90β was immunoprecipitated using anti-HA antibody, and then p14ARF, CHIP, and HSP90 were detected using antibodies as indicated. B, Cells were transfected with plasmids as indicated. After 24 hours, the cell lysates were immunoprecipitated using anti-HA or anti-FLAG antibody. C, GST pull-down assay was performed using in vitro translated p14ARF and purified GST-CHIP. D, HeLa (human cervical cancer) cell lysates were immunoprecipitated using anti-p14ARF, anti-CHIP, and anti-HSP90α/β antibodies. E, Plasmids expressing Myc/full-length p14ARF, Myc/p14ARF deletion mutants, and HA-CHIP were transfected as indicated. The cell lysates were immunoprecipitated using anti-Myc antibody. F, Plasmids expressing Myc/full-length CHIP, Myc/CHIP deletion mutants, and p14ARF-FLAG were transfected as indicated. The cell lysates were immunoprecipitated using anti-Myc antibody. G, Constructs were transfected as indicated. The cell lysates were immunoprecipitated using anti-FLAG antibody. p14ARF protein expression was adjusted for comparing interactions with identical amounts of p14ARF. Asterisks, chains of antibodies.