FIGURE 5:

Hsc70 binding is required for CFTR ubiquitination. (A) Microsomes containing newly synthesized CFTR were subjected to an in vitro degradation assay in the presence of 20 μM CBag and 100 μM MG132 as indicated. At T = 1 h, aliquots were immunoprecipitated with anti-(mono-/poly-) ubiquitin antibody (FK2) or NIS and analyzed by SDS–PAGE and phosphorimaging. Right gel shows 25% input of sample before immunoprecipitation. (B) Following CFTR synthesis, hexokinase (lanes 1 and 2), buffer (lane 3), or His-CBag (lane 4) was incubated with translation reaction for 10 min. CFTR was then solubilized and pulled down with immobilized GST (lane 1) or GST-CHIP (lanes 2–4). (C) Isolated microsomes containing newly synthesized CFTR were incubated with or without CBag and ATP for 1 min to release Hsc70 and immediately solubilized in Triton X-100. Samples were incubated with immobilized GST(lane 1) or GST-CHIP (lanes 2–4). Bottom gels (B and C) show CBB staining of GST proteins.