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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma

Fig. 5

FAM289 protein localized in nucleus could activate ERK signaling pathway depending on its interaction with Galectin-1. (A) Galectin-1 could affect the intracellular localization of FAM289 protein and facilitate its entry into the nucleus. HEK293T and U251 cells were transfected with RFP-galectin-1 and GFP-FAM289 alone or together and stained with DAPI. Scale bars, 40um. (B) Cytoplasmic or nuclear fractions from HEK293T cells transfected with Flag-Galectin-1plasmid or GFP-FAM289 plasmid were immunoblotted with anti-Flag or anti-GFP. Purity of the nuclear and cytoplasmic fractions was verified by immunoblotting using anti-H3 or anti-GAPDH. (C) FAM289 and Galectin-1 mainly interact with each other in the nucleus. Cytoplasmic or nuclear fractions from HEK293T cells transfected with Flag-Galectin-1, GFP-FAM289 or both plasmids were extracted and Co-IP experiment was performed using anti-Flag antibody. (D) pEGFP-FAM289, pEGFP-FAM289-NLS, pEGFP-FAM289-NES and control vectors were transfected into U251 cells. These cells’ proliferation was analyzed by xCELLigence RTCA. (E) The cell cycle was analyzed by flow cytometry after PI staining. The data were processed with ModFit LT program. (F) pEGFP-FAM289, pEGFP-FAM289-NLS, pEGFP-FAM289-NES and control vectors were transfected into U251 and U87-MG cells. The lysates were immunoblotted with anti-ERK, anti-pERK, anti- DNMTS and anti-CD133 antibodies respectively

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