Figure 1.

ERdj3 binds to γHC directly. (A) COS-1 cells were co-transfected with cDNAs encoding γHC and the indicated HA-tagged ERdj3 constructs. Metabolically labelled, crosslinked cell lysates were immunoprecipitated with anti-HA or Protein A Sepharose alone. Isolated proteins were separated by reducing SDS–PAGE. (B) Ag8.8 cells were metabolically labelled for 16 h with [³⁵S]methionine and cysteine and incubated with (lane 1) or without (lanes 2 and 3) DSP. Cell lysates were prepared with (lane 2) or without (lanes 1 and 3) ATP and immunoprecipitated with Protein A Sepharose. (C) Wild-type (WT) and QPD mutant (Mut) ERdj3 were in vitro translated and run directly (lanes 1 and 2) or incubated with the free γHC immobilized on Protein A Sepharose beads (lanes 4 and 5) prepared as in lane 2 in Figure 1B or with Protein A beads alone (lanes 6 and 7).