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Figure 1


Fig. 1. Strategy for interfering with 14-3-3-dependent surface expression of membrane proteins. (A) Schematic diagram of the fusion protein employed to sequester 14-3-3 proteins and (below) sequence of the R18 peptide. (B) Endogenous 14-3-3 protein in Xenopus oocytes is associated with the scavenger protein pGpLI-R18. Western blots of 14-3-3 and pGpLI-R18 in cellular extracts (extract) from uninjected or pGpLI-R18-injected oocytes, and in the flow-through (IgG flow-through) and eluate (IgG eluate) after incubation of the extracts with IgG-Sepharose. Gels were probed with an anti-14-3-3 ß antibody detecting all isoforms: the pGpLI-R18 protein is visible because of its protein-G moiety. M indicates molecular mass in kDa. (C) Surface expression of KCNK3, ß2 adrenergic receptor and Kir2.1 measured in the absence (black bars) and presence (grey bars) of the 14-3-3-scavenger pGpLI-R18. Data are normalized to the values measured in the absence of pGpLI-R18. KCNK3PC, n=35 (six batches of oocytes); ß2 adrenergic receptor, n=15 (three batches of oocytes); Kir2.1HA, n=13 (three batches of oocytes). Only for KCNK3 did 14-3-3 sequestration significantly reduce surface expression (P<0.0005).





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