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. 2016 Oct 10;113(43):E6610–E6619. doi: 10.1073/pnas.1605350113

Fig. 10.

Fig. 10.

V-1 is phosphorylated on Ser-22 in vivo, and a phosphomimetic version of V-1 interacts weakly with CP in vitro. (A) Western blot of extracts of WT cells (lane 1) and V-1–null cells (lane 2) that were resolved on urea-glycerol gels and probed with the antibody to D.d. V-1. (B) Western blot of extracts of WT cells that were not (lane 1) or were (lane 2) treated with phosphatase before loading on urea-glycerol gels and probed with the antibody to D.d. V-1. (C) Western blot of the material eluted from anti-FLAG M2 beads that had been incubated with lysates of cells expressing FLAG-V-1 and that were not (lane 1) or were (lane 2) treated with phosphatase before loading on urea-glycerol gels and probed with the antibody to D.d. V-1. (D) Western blot of the material eluted from resins that had been loaded with equal amounts of GST-WT V-1 (lane 1) or GST-V-1 S22E (lane 2), incubated with cell extracts, and washed. The blot was probed with an antibody against D.d. CPβ. (E) Representative pyrene-based, seed-initiated, actin polymerization assays performed in the presence of 7 nM CP and increasing amounts of GST-V-1 S22E added before seed initiation (see color-coded key). (F) Mean and SDs from three independent assays performed as in E and plotted as the fraction of capped filaments vs. V-1 concentration.