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. 2021 Mar 17;7(12):eabe2470. doi: 10.1126/sciadv.abe2470

Fig. 2. GREB1 GT activity is important in stabilizing ERα.

Fig. 2

(A) FLAG pulldown was analyzed by Western blot in MCF7-WT or GREB1-KO cells expressing FLAG-ERα. (B) Catalytically dead mutant GREB1 (GT-Mut) was generated by mutating indicated residues in GREB1’s GT domain. (C) The growth of GREB1-KO cells transduced with Vec, GT-WT, or GT-Mut was assessed by colony formation assay with crystal violet staining. Photo credit: Sultan Abda Neja, Institute of Cell and Molecular Biology, A*STAR. (D and E) GREB1-KO cells transduced with GT-WT or GT-Mut were subcutaneously injected to the flanks of the same NOD/SCID mice, each cell line on one side. The resulting tumors in these mice were harvested (D) and weighted (E). n = 8; ****P < 0.0001 by unpaired t test. Photo credit: Anandhkumar Raju, Institute of Molecular and Cell Biology, A*STAR. (F) GREB1-KO cells transduced with Vec, GT-WT, or GT-Mut. (G) ERα, GT-WT, and GT-Mut were individually purified from 293T. In vitro assay using a UDP-Glo Glycosyltransferase Assay kit was performed as indicated. n = 3. (H) S. cerevisiae yeast was transfected with either Vec or FLAG-conjugated GREB1 construct.