(A) Immunoblot of Xenopus eggs fractionated over a sucrose gradient and probed with importin α and beta-catenin antibodies. Importin α is found in the cytoplasm and also cofractionates with beta-catenin as a marker for plasma membrane mostly in the heavy membrane fraction.
(B) Immunoblot of cytoplasm and membrane fractions isolated from stage 3 and stage 8 embryos and probed with importin α and beta-catenin antibodies. A greater fraction of membrane-associated importin α is observed at stage 8.
(C) Blot of immunoprecipitated recombinant wild-type and mutant importin α proteins (2S: S154A, S490A; NP: S154A, S490A C230A, C454A) retrieved from Xenopus egg extract and probed with importin α antibodies or streptavidin to quantify incorporation of biotin-labeled palmitate (Martin, 2013).
(D) Blot of immunoprecipitated recombinant importin α retrieved from Xenopus egg extract following a 1 hour incubation with DMSO solvent, 10 μM palmostatin, or 1 μM Wnt-C59 and probed with importin α antibodies and streptavidin to quantify incorporation of biotin-labeled palmitate (Martin, 2013).
(E) Fluorescence images of GFP-tagged wild-type or NP mutant importin α added to Xenopus egg extract, co-stained with FM 4–64X to visualize plasma membrane lipid derived vesicles, in the presence of DMSO control or drugs that alter palmitoylation. Two vesicles are shown for each condition. GFP-importin α wt localization to vesicles was enhanced by Palmostatin and inhibited by Wnt-C59, whereas GFP-importin α NP did not co-localize with plasma membrane lipid vesicles under any condition. Scale bar, 10 μm.