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Fig. 2 | Cell & Bioscience

Fig. 2

From: Drosophila Prominin-like, a homolog of CD133, interacts with ND20 to maintain mitochondrial function

Fig. 2

Prominin-like interacts with ND20 proteins. a Schematic illustration of truncated Prominin-like region. The cDNA fragments encoding amino acid: 55–164, 236–491, 558–851, and 875–1014 of prominin-like were cloned into vectors for interaction assays. b, c Yeast two-hybrid assays: pGBKT7-53 and pGADT7-T were used as positive controls with pGBKT7-Lam and pGADT7-T as negative controls; Mated yeast strains containing both bait vectors (pGBKT7-PL-1,-2,-3 or -4) and prey vectors (pGADT7-ND20) were plated on double dropout (DDO/X) (b) and higher stringency quadruple dropout (QDO/X/A) selective agar medium (c); there were one-to-one correspondences among af in b and c. d GST pull-down assays. GST or GST-PL-1 fusion protein generated by E. Coli BL21 cells was purified by glutathione agarose resin, followed by incubation of the resin with HA-ND20 protein expressed in S2 cells. After washing with PBS, the bound proteins were analyzed by Western blotting with anti-HA antibodies. e, f Co-IP assay analysis of the interaction between Prominin-like and ND20. Cells were co-transfected with pAC5.1-Flag-Prominin-like and pAHW-HA-ND20 in S2 cells; co-transfection of pAC5.1-Flag and pAHW-HA served as negative controls. Some of the lysates were subjected to input assays to assess β-tubulin, Flag-Prominin-like and HA-ND20 protein levels, and the remainder were subjected to IP assays. e HA-ND20 proteins were detected by Western blotting using anti-HA antibodies. f Flag-prominin-like proteins were detected by Western blotting using anti-Flag antibodies in the presence of the cross linker, DTSSP

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