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. 2023 May 19;42(13):e112504. doi: 10.15252/embj.2022112504

Figure 6. Loop‐proximal Ndc80‐Ndc80 crosslinking rescues increased diffusivity of loopless Ndc80 trimers.

Figure 6

  1. Representation of the peptides used to raise AB‐849 and AB‐850 and immunoblots showing their recognition of wild‐type, M5 and loopless Ndc80 complexes. 9G3 is a commercially available monoclonal antibody raised against NDC8056–642, later shown to recognize NDC80200–215. Asterisk shows the non‐specific recognition of another protein, presumably NUF2.
  2. Diffusion of full‐length and loopless Ndc80 trimers in absence and presence of AB‐849. The primary rabbit polyclonal AB‐849 was detected using a Alexa650‐labeled anti‐rabbit secondary IgG antibody. Scale bars: vertical (100 s), horizontal (5 μm).
  3. One‐dimensional diffusion of full‐length (blue), loopless (orange), and M5 (black) Ndc80 trimers in presence and absence of AB‐849 and AB‐850 as described in panel (B) (see Appendix Fig S4 for more information). Traces of Ndc80 trimers (with n indicated in the legend for panel D) on microtubules were analyzed. Traces were split into segments of 60 s and averaged (see Materials and Methods). Mean (circles) and SEM (shaded areas) values are shown. We note that the omission of reducing agents, a precondition to use the antibody as a crosslinker, slightly decreased the overall diffusion of Ndc80‐modules on microtubules (compare with Fig 2E).
  4. Summary showing diffusion coefficients (μm2/min) that follow from the data shown in panel (C). Mean values, SEM, and number of diffusion traces (n) are indicated. Statistically significant differences were determined using a two‐tailed t‐test. P‐values: no AB, FL vs. ΔL: 0.0180 (*); ΔL, no AB vs. AB‐849: 0.0108 (*); M5, no AB vs. AB‐849: 0.0458 (*); ΔL, AB849 vs. AB‐850: 0.0156 (*); AB‐850, ΔL vs. FL: 0.0461 (*); AB‐850, ΔL vs. M5: 0.0494 (*).

Source data are available online for this figure.