Figure 4

Hook2 expression has no effect on the biochemical properties of CFTR. (A) Vero cells transfected with ΔF508-CFTR were treated (+) or not (-) with lactacystin (lact), lysed and immunoblotted (blot:) for CFTR (left) and ubiquitin (middle). Stars indicate bands specifically seen in ΔF508-CFTR-expressing cells, compared to untransfected controls (-). As shown previously [39, 40] the upper band of CFTR runs at the same position as the ubiquitin-rich band in ΔF508-CFTR transfected and lactacystin treated cells. To control for loading membranes were stained with MemCode after immunoblotting (right). (B) Anti-ubiquitin antibodies also recognized the upper band of immunoprecipitated CFTR. (C) CFTR was expressed in the presence of lactacystin and analyzed by differential detergent extraction. Little CFTR was extracted by 1% Triton X-100 (1). Whereas the lower band of CFTR is soluble in 1% SDS (IS^S), a fraction of the upper band is detected in the pellet of the 1% SDS extract (IS^P) and needed to be solubilized in 6% SDS, as previously observed for other proteins in aggresomes [4, 14]. (D) CFTR western blot of whole cell homogenates of untransfected cells (lane 1) or cells expressing ΔF508-CFTR-GFP alone (lane 2) or together with hook2 (lanes 3 and 4 are duplicates) showed that hook2 had no effect on the migration of ΔF508-CFTR in SDS-PAGE gels. The apparent reduced level of CFTR-GFP after hook2 co-expression was not consistently observed. (E) Hook2 co-expression did not significantly change the fraction of ΔF508-CFTR detected in Western blots of the 0.1% or 1% Triton-X 100 soluble or insoluble (IS) fractions.