Partial loss of Knl1, RZZ, and Mad1 at Zwint1-depleted kinetochores. (A) Immunoblot of endogenous Zwint1 and α-tubulin (loading control) from HeLa cells treated with control or Zwint1 siRNA for 48 h. (B, E, F, H, and I) Nocodazole-treated HeLa cells treated with either control luciferase or Zwint1 siRNA were immunostained using anti-Hec1 9G3 (B, E, F, and H) or anti-Zwint1 (I) and either anti-Zwint1 (B), anti-Knl1 (E), anti-Rod (F), anti-Zwilch (H), or anti-Mad1 (I) antibodies. (C, D, G, and J) Kinetochore fluorescence was normalized relative to Hec1 (C, D, and G) or ACA (J) in control and Zwint1-depleted cells. n = 100 kinetochores; P < 0.001. Error bars are SD from the means. Bars, 5 µm.