Xenbase Image ID: 49171

Fig. 3. Immunolocalization of Xenopus Kir6.1 and SUR2A. A) Western blotting with anti-myc and anti-Kir6.1 antibodies against lysates from Stage 11 embryos injected at one cell with xKir6.1-myc mRNA. The blot confirms that both antibodies recognize products with molecular weights at multiples of 60 kDa (lanes 2 and 5). Although this is slightly larger than the up to ~ 51 kDa size recognized for Kir6.1 in other studies ([Foster et al., 2008] and [Suzuki et al., 1997]), it runs at the same size as the Kir6.1-myc construct probed with anti-Myc (blue arrow, lane 2), confirming that the anti-Kir6.1 antibody recognizes xKir6.1 protein. The higher molecular weight bands were not observed in lysates from uninjected embryos (lanes 1 and 4). Lysates of uninjected one-cell Xenopus embryos shows that 1-cell embryos contain maternal xKir6.1 protein (lanes 4 and 7, blue arrow). Pre-incubation of the antibody with Kir6.1 peptide (lane 8), but not with a Kir6.2 peptide (lane 9), abolished immunoreactive bands. Anti-myc recognized a faint band at about 45-kDa in uninjected embryos (lane 1); this may be endogenous c-myc. B) A SUR2 antibody recognizes several bands in one-cell Xenopus lysates, including a 130 kDa band, approximately the size of SUR2 (150 kDa). C–K) Immunofluorescence with anti-Kir6.1 antibody on sections from early to late cleavage stage in Xenopus embryos. C) Control sections without primary or (D) secondary antibodies exhibit low level staining and low levels of yolk autofluorescence. E) Positive control anti-H/K-ATPase antibody shows expected left–right asymmetric localization. F, G) At two to four cell stage, xKir6.1 exhibits low levels of expression and indistinct localization. H) At Stage 6, xKir6.1 is localized to intracellular domains (green arrows) and to isolated regions on plasma membranes facing the blastocoel (green arrowheads). I, J) At stage 7, Kir6.1 is localized to intracellular domains (I, green arrows) and membranes on the basal face of animal cap cells lining the blastocoel (J, green arrowheads). Intercellular punctate staining is also observed (J, blue arrows). K) At Stage 8, xKir6.1 remains localized to intracellular domains (K, green arrows) and to plasma membrane in many parts of the embryo (K, green arrowheads). L–N) When intracellular domains recognized by anti-Kir6.1 (L) are co-localized with nuclear DNA (M), they appear to lie peripheral to the nucleus (N), suggestive of endoplasmic reticulum localization. O–R) Immunofluorescence with anti-SUR2A antibody in early cleavage Xenopus embryos show punctate, tight junction-like staining at from two cell to stage 32/64 cell. Q′ is a close up of the staining at the junction of a cleaving two to four cell embryo, indicated with a white arrow in Q. Image published in: Aw S et al. (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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