Figure 6.

Hslo fluorescent fusion proteins do not form insoluble aggregates in CHO cells. Western blot of CHO cells transfected with wild-type hslo (WT, top panel), a construct expressed at the plasma membrane, 1062-YFP (1062; second panel), a construct that is retained intracellularly, 780-YFP (780; third panel), or nontransfected cells (NT; bottom panel). Cells were permeabilized in lysis buffer containing 0%, 0.1%, 0.4%, 1%, and 2% Triton X-100 detergent. S, soluble fractions; I, insoluble fractions. For each construct, essentially all of the hslo protein is solubilized by 1% detergent. Doublet bands were sometimes seen from the fusion proteins. These were unaffected by removal of N-linked glycosylation after PNGase F pretreatment, but became single bands when higher SDS concentrations were used. Presumably they arose from incomplete unfolding of GFP domains in SDS. Hslo bands were immunodetected with anti-FLAG monoclonal antibodies. Bars to the left of the panels indicate migration of molecular mass markers (75, 100, and 150 kD).