Figure 5. Effect of Truncated Receptor Expression on the Amount of Full-Length M6P/IGF2R in Whole-Cell Lysates vs. Plasma Membranes.

HEK 293 cells were transfected with pCMV5 vector or the cDNAs (10 μg) encoding Rep9F or Rep14 alone or by co-transfection with empty vector or 10 μg of cDNA encoding the full-length WT-M receptor, as indicated. Transfected DNA was balanced to a total of 20 μg per dish in all cases. Aliquots (50μg protein) of A) detergent extracts (lysates) and B) plasma membranes were analyzed by immunoblotting with the antibodies indicated to the right of each panel. Arrows designate the various forms of the receptor. B, bottom panel shows a portion of the plasma membrane blot stained with a solution of 0.1% Ponceau S in HBS; the intensity of an irrelevant 39K band serves as a loading control. One-way ANOVA of the data from three replicate experiments indicated that differences in intensity of the full-length bands were not significant when comparing Rep9F-transfected vs. pCMV5 vector or Rep9F co-transfected with WT-M controls. The effect of Rep14F on intensity of the endogenous receptor bands and the WT-M exogenous receptor bands was significant (P <0.025) in both lysates and plasma membranes.