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Fig. 4. C-terminal residues other than RKR contribute to 14-3-3 recruitment by the Kir6.2 tail. (A) Sequence of the WT, 
C10 and C16 Kir6.2 C-terminal tails used in the reporter constructs. (B) Coomassie-Blue-stained gels demonstrating equal loading of the Kir6.2 reporter proteins. The immobilized bait protein was eluted using SDS-PAGE sample buffer, resolved by electrophoresis and stained using Coomassie Blue. M indicates molecular mass in kDa. (C) HeLa cell cytosol (2% input) and eluates after incubation of cytosol with IgG-Sepharose preloaded with tetrameric protein A fusions (pApLI) of the indicated Kir6.2-derived tails (bait proteins, see B). RKR is the full-length WT sequence, KKK and AAA (Michelsen et al., 2006; Yuan et al., 2003; Zerangue et al., 1999) are inactive variants of the RKR motif (KKK preserves the charge), C10 and C16 are the C-terminal deletions (see A), RKR_P-A (KFSISADSLS), RKR_PL-GG (KFSISGDSGS) and RKR_S-A (KFAIAPDALA) are mutant variants of the last ten residues of Kir6.2 (pertinent sequence in parentheses, bold characters indicate mutated residues). (Top panel) Coomassie-Blue-stained gel of the eluates (80%). (Bottom panel) Western blot analysis of the same samples (10%). The membrane was probed with a pan-reacting antibody raised against the ß isoform of 14-3-3 proteins. The lowest panel combines the results for the RKR_S-A variant. The asterisk indicates a band that was not detected by western blotting against 14-3-3. To confirm that the band did not contain 14-3-3, e.g. in a modified form, we analyzed the protein by mass spectrometry. This revealed that the band contains p32/C1QBP, the precursor of a strongly acidic protein destined for the mitochondrial matrix (Dedio et al., 1998; Muta et al., 1997). The results for all constructs were very similar in four independent experiments.
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