Figure 1.

Expression of bicistronic SplitΔR_pNUT, SplitΔR_pIND, and SplitΔR_pIND+RD_pNUT CFTR in BHK cells. (A) Western blots of cell lysates from six BHK variants that had been transfected with SplitΔR_pNUT CFTR and selected using 500 μM methotrexate. Note the expression of both amino-terminal (aa 1–634; top) and carboxy-terminal (aa 837–1480) halves of CFTR in colonies 1, 2, 4, and 5, as detected using the monoclonal antibodies L12B4 or M3A7, respectively. (B) Effect of glycerol treatment on maturation of full-length wild type (WT) or SplitΔR CFTR. Mature CFTR was sensitive to PNGase but not EndoH, consistent with the complex glycosylation (Band ‘C'). The back half of SplitΔR was sensitive to EndoH, indicating the presence of core oligosaccharide chains, which are characteristic of immature (Band ‘B') CFTR. (C) Western blot probed with antibody against front (L12B4) and back (M3A7) half-molecules showing the effect of incubating cells overnight with 10% glycerol at 26°C on expression of SplitΔR_pNUT and wild-type CFTR. (D) Inducible expression of SplitΔR_pIND with or without coexpression of RD_pNUT. Western blot showing the front, back, and R domain (450) with (+) or without (−) induction by 10 μM Ponasterone A (Pon. A) for 24, 48, or 72 h. (E) Covalent crosslinking of coexpressed R domain and SplitΔR fragments into a complex. Cells were incubated with 2 mM dithiobis (succinimidyl propionate) (DSP) for 30 min, lysed, and immunoprecipitated using the anti-R domain. After crosslinking, most R domain (23 kDa) migrates in a high-molecular-weight complex of 175–200 kDa that also contains the front and back halves of SplitΔR. (F) Cell surface biotinylation of SplitΔR_pIND, SplitΔR_pIND+RD_pNUT, or wild-type CFTR. Both halves were pulled down on streptavidin beads after cell surface biotinylation using sulfo-NHS-SS-biotin (left and right panels, respectively). SplitΔR was not co-precipitated if biotin was omitted from the reaction mix (‘no biotin').