ALIX and ESCRT-I constitute parallel CHMP4B recruitment arms. (A) Confocal images showing CHMP4B at the cytokinetic bridge of HeLa cells stained for DNA (Hoechst), α-tubulin, CHMP4B, and the midbody marker RacGAP1 upon siRNA treatment as indicated. Bars, 5 µm. (B) Quantification of CHMP4B localization at the midbody (error bars indicate SEM; n > 30 cells from three independent experiments; unpaired t test; ***, P < 0.001). (C) Schematic view of the CHMP4B constructs used in D and E. (D) Confocal images of HeLa cells stably expressing CHMP4B-V5 or CHMP4B^ΔALIX-V5 stained for DNA (Hoechst), α-tubulin, V5, and MKLP1 upon siRNA treatment as indicated. Bars, 5 µm. (E) Quantification of the CHMP4B or CHMP4B^ΔALIX signal at the midbody in cells stably expressing CHMP4B-V5 or CHMP4B^ΔALIX-V5 (error bars indicate SEM; n = 15 cells from three independent experiments; unpaired t test; **, P < 0.01). Knockdown of TSG101 and ALIX is shown by Western blot. (F) Live imaging of CHMP4B enrichment at the midbody relative to CEP55 in HeLa cells stably expressing mCherry-CEP55 and CHMP4B-eGFP (error bars indicate mean with 95% confidence interval; n ≥ 27 cells from five independent experiments; control siRNA 35 ± 23.2 min, ALIX siRNA 72 ± 51.4 min, TSG101 siRNA 71 ± 34.7 min (± SD); mixed factor model; **, P ≤ 0.01). (G) Schematic view of cytokinetic CHMP4B recruitment.