The 3′ UTR is required for CHMP4B translation at the MB during telophase. (A) HeLa cells were transfected with RFP-CHMP4B-3′UTR and plated on glass-bottom dishes. The accumulation of RFP-CHMP4B at the MB during telophase was then imaged using time-lapse microscopy for 120 min with 15 min time-lapse. Asterisk marks the midbody. Scale bars: 10 μm. Inset scale bars: 1 μm. (B) HeLa cells stably expressing GFP-MKLP1 were transfected with either HA-CHMP4B-3′UTR (images on the left) or HA-CHMP4B (images on the right). Cells were then fixed and incubated with anti-HA and anti-puromycin antibodies followed by proximity ligation assay (PLA). Asterisk marks the midbody. Boxes mark area shown in higher magnification insets. Scale bars: 10 μm. Inset scale bars: 1 μm. (C) Negative controls for the PLA experiment are shown in B. Cells in the image on the left were not treated with puromycin but transfected with HA-CHMP4B-3′UTR. Cells in the image on the right were treated with puromycin but not transfected with HA-CHMP4B-3′UTR. Asterisk marks the midbody. Scale bars: 10 μm. (D) Quantification of the levels of PLA signal in the midbody area. Data shown are the means and SEM derived from all cells analyzed. Each circle indicates the data derived from one dividing cell.