Fig. 3

Expression of a phospho-mimetic mutant of Eg5 rescues defects in NEK7-depleted neurons. a Extracts from 14 DIV neurons infected at 7 DIV with lentivirus containing pLL3.7 plasmids that express either GFP, FLAG-Eg5 wild-type, or S1033 mutants. Lysates were probed with Eg5 and FLAG antibodies by western blotting. α-Tubulin was used as loading control. b Neurons, infected at 7 DIV with control or Nek7 shRNA virus, and co-transfected with FLAG-GFP or FLAG-Eg5 (wild type or mutants as indicated) and GFP plasmid, were fixed at 14 DIV and co-stained with GFP antibody (shown in the figure) and with FLAG antibody (for selecting cells with simillar levels of FLAG-Eg5 expression). Scale bar, 50 μm. c Quantification of total dendrite length in 14 DIV neurons as in (b). d Sholl analysis of 14 DIV neurons as in (b). Mean number of intersections are plotted; n = 3 independent experiments. Total number of neurons: 30 (shControl+FLAG-GFP), 33 (shNek7+FLAG-GFP), 27 (shNek7+FLAG-Eg5), 22 (shNek7+FLAG-Eg5 S1033A), and 24 (shNek7+FLAG-Eg5 S1033D); n.s. non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed t-test. e Representative images of dendritic spines in primary dendrites of anti-GFP-stained neurons as in (b). Scale bar, 1 μm. f Quantification of the density of dendritic spines as in (d); n = 3 independent experiments, total of 11 to 17 neurons per condition, total number of spines analyzed per condition 292 < n < 667. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed t-test. Error bars: s.e.m. Columns in all graphs show means and dot overlays individual data points