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Figure 2
Construction and characterization of vPIP mutants. (a) Schematic diagram of vPIP deletion and point mutants. Two deletion mutants, vPIP ΔN (deletion of amino acids 2–26) for Nβ and α1 and vPIP ΔC (deletion of amino acids 277–301), and two point mutants, vPIP mI (F5A, S12A and T16A) for interaction sites and vPIP mP (H47A and D50A) for pore sites, were constructed. An additional mutant containing both mI and mP was also generated (vPIP mIP). (b) The structure of vPIP mutations with positional indicators. The positions of the mutations are marked in the monomer structure of vPIP. (c) Expression of vPIP mutants. FLAG-tagged vPIP mutant constructs were transfected into HeLa cells. After 24 h, expression of the vPIP mutants was analyzed by Western blotting. (d) Subcellular localization of vPIP mutants. HeLa cells were transfected with the GFP-tagged vPIP mutants, fixed at 24 h post-transfection and immunostained with anti-PARP-1 antibody. The nuclei were stained with DAPI (blue). The scale bar is 20 µm in length. (e) Dimerization of vPIP mutants. MYC-tagged vPIP mutants were co-transfected with FLAG-tagged vPIP mutants into HEK293T cells for 48 h. The cells were harvested and subjected to co-IP assays using anti-FLAG. The results were analyzed by Western blotting.

IUCrJ
Volume 5| Part 6| November 2018| Pages 866-879
ISSN: 2052-2525
https://doi.org/10.1107/S2052252518013854