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. 2017 Mar 23;7:44863. doi: 10.1038/srep44863

Figure 2. Identification of WAVE2 domains and lysine residues required for its ubiquitylation.

Figure 2

(a(i)) Schemes of YFP-WAVE2 wt and YFP-WAVE2 deletion mutants. WHD, WAVE homology domain; B, basic region; PRD, proline-rich domain; VCA, verprolin-homology cofilin-homology acidic domain. (a(ii)) 293 T cells were co-transfected with constructs encoding HA-tagged ubiquitin together with YFP-WAVE2 wt or with YFP-WAVE2 mutant forms as indicated. YFP-WAVE2 was immunoprecipitated using anti-GFP antibody; complexes were resolved by SDS-PAGE and immunoblotted for ubiquitin using anti-HA antibody, and for YFP-WAVE2 using anti-GFP antibody. Ubiquitylated YFP-WAVE2 wt appears at a MW of ~110 kDa, which is 8 kDa above the MW of YFP-WAVE2 (~102 kDa). Data shown are representative of at least three independent experiments. (b) 293 T cells were co-transfected with constructs encoding HA-tagged ubiquitin together with YFP-WAVE2 wt or with YFP-WAVE2 mutant forms as indicated. Cells were lysed and immunoprecipitated with anti-GFP. IPs were separated by SDS-PAGE, transferred, and blotted with anti-HA and anti-GFP antibodies. Data shown are representative of three independent experiments. (c(i)) Schematic illustration of WAVE2 structural domains. The WAVE2 WHD domain contains ten lysine residues, as indicated. (c(ii)) Schemes of YFP-WAVE2 deletion mutants. (c(iii)) 293 T cells were co-transfected with constructs encoding HA-tagged ubiquitin together with YFP-WAVE2 wt or with YFP-WAVE2 mutant forms described in cii. Cells were lysed and immunoprecipitated with anti-GFP. IPs were separated by SDS-PAGE, transferred, and blotted with anti-HA and anti-GFP antibodies. Data shown are representative of at least four independent experiments. (d) 293 T cells were co-transfected, immunoprecipitated, and blotted as described above. Densitometric analysis of the bands presented was performed using ImageJ and normalized to the GFP densitometry values. Error bars show standard error of the mean (SEM). Significance was determined by two-tailed Student’s t-test and is indicated by asterisks (*P < 0.05). Data shown are representative of at least three independent experiments.