Figure 4.

Calnexin interactions are required for efficient assembly of MSD1 and MSD2. (A and C) Assembly of CFTR membrane spanning domains. HEK293 cells were transfected with CFTR 1-837 or CFTR 837-1480, and cells were either treated 5 h posttransfection with 10 μg/ml BFA or 5 mM CAS, or 18 h posttransfection with 200 μM ALLN where indicated. Cells were harvested 24 h after transfection, and cell lysates subjected to Western analysis with CFTR N-terminal or NBD2 specific antibodies. Maturely glycosylated 837-1480 is indicated as C band, and immaturely glycosylated 837-1480 is indicated as B band. Tubulin is used as a loading control. (B) HEK293 cells were transfected with pcDNA CFTR 1-837 and pcDNA CFTR 837-1480, and harvested 24 h after transfection. Cells were then lysed in 1% SDS with 0.1 mM B-mercaptoethanol, sonicated, and incubated at 37°C for 10 min. Samples were then diluted fivefold into reaction buffer (5% Triton, 100 mM sodium phosphate, pH 5.5, for Endo H or 100 mM sodium phosphate, pH 7.6, for PNGaseF), the indicated enzyme (PNGaseF or EndoH) was added, and samples were incubated overnight. Sample buffer was added to a final 1× concentration, and samples were run on 10% SDS-PAGE gels. Western blots were performed with the α-NBD2 CFTR antibody. Bands B and C represent the immature and maturely glycosylated forms, respectively, and band A represents the nonglycosylated form of CFTR 837-1480. (D) HEK 293 cells were transfected with the calnexin shRNAmir construct or the nonsilencing control as indicated in Materials and Methods. Cell lysates were subjected to Western Blot analysis with the indicated antibodies.