Fig. 4. In vitro MT nucleation activity on BY-2 nuclei in control and inhibition assays. (A) Without the antibody, the relative nucleation activity was 100%, and this was compared with the observed after incubation in the presence of anti--tubulin 1/200 (B₁), 1/100 (B₂), 1/50 (B₃); anti-Spc98pA 1/200 (C₁), 1/50 (C₂); anti-Spc98pB 1/200 (D₁), 1/50 for 20 minutes (D₂), 40 minutes (D₃), 60 minutes (D₄); non-specific anti-Spc98pC 1/200 (E) and anti-GFP 1/200 (F). A competition assay incubating anti-Spc98pA 1/200 with the corresponding peptide 1µM for 1 hour before nucleation is shown (G). 70 to 95% inhibition of MT nucleation was observed using specific antibodies, indicating that both Spc98p and -tubulin are directly involved in MT nucleation.