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. 2020 May 29;9(6):1355. doi: 10.3390/cells9061355

Figure 4.

Figure 4

C-terminal truncation of CYFIP prevents WRC assembly and lamellipodia formation. (A) Cell morphology of CYFIP1/2 KO cells (clone #3) expressing EGFP-tagged WT or E1150Dfs*3 CYFIP1 mutant, leading to a C-terminally, truncated protein. Scale bar, 10 µm. (B) Quantification of lamellipodia formation, done as described for Figure 1A. Note that CYFIP1 WT control is as in Figure 1A. (C) Western blotting of B16-F1 cells or CYFIP1/2 KO cells (clone #3) transfected with indicated constructs and probed for expression of CYFIP and WAVE. (D) B16-F1 cells were transfected with indicated constructs, lysed and subjected to immunoprecipitation analysis to assay interaction of the E1150Dfs*3 mutant of CYFIP1 versus WT or R87C-CYFIP1 with WRC subunits WAVE, Nap1 and Abi-1. (E) Cycloheximide treatment of CYFIP1/2 KO cells (clone #3) transfected with either WT- or E1150Dfs*3-CYFIP1. Cells were treated with 20 µg/mL cycloheximide for indicated times (hours), lysed and cell extracts subjected to western blotting against CYFIP1 (top). The decay of protein expression was plotted over time and derived protein half-life estimated. One representative experiment out of two is shown.