Extended Data Fig. 6: GS directly interacts with NUP88 through a charge-charge interaction.
From: Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth
a, Surface electrostatic potential of the crystal structure of GS (PDB code: 2QC8) showing the two charged patches for potential protein-protein interaction. The surface electrostatic potential was calculated in PyMOL. b, A schematic showing the design of GS-del6 mutant in which the first six amino acids of the K103-R119 motif are deleted. c, Co-IP experiments were conducted with the lysate of 293T cells transfected with V5-tagged NUP88 and FLAG-tagged GS-WT or GS-del6 using anti-FLAG antibody. d, Left: In vitro pull-down experiments were conducted with the mixture of purified V5-NUP88 and FLAG-GS-WT or FLAG-GS-m4 protein using anti-FLAG or anti-V5 antibodies; right: Western blot showing V5-NUP88, FLAG-GS-WT and FLAG-GS-m4 protein purified from 293T cells transfected with V5-NUP88 and FLAG-GS expressing constructs. e, Silver staining for purified FLAG-GS-m4 proteins. f, Western blot showing the expression of GS in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-m4. g, Quantification of the duration of mitotic progression of H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-m4 in the presence of 2 mM glutamine. (n=60 individual cells for each group of cells). Data from mitotic analysis were plotted in the format of a violin plot. Unpaired, two-tailed t-tests were applied for g. *: p<0.05, **: p<0.01.
