(A) Schematic representing the organisation of PKCε, with the residues of the Inter-C1 Domain (IC1D) noted with the three residues that make up the actin binding motif in red, and the two phosphorylation sites in blue and green. (B) Representative immunofluorescence images of dividing HEK293T cells expressing GFP-PKCε, GFP-PKCε-D532N, GFP-PKCε-D532N-L223A-K224A-E227A, GFP-PKCε-D532N-T228A or GFP-PKCε-D532N-S234A. Cells stained for DNA (blue) and tubulin (red), with GFP-PKCε shown in grey; the midbody region highlighted is expanded for each image. Scale bar = 20 µm. (C) Quantitation of dividing cells with GFP-PKCε, GFP-PKCε-D532N, GFP-PKCε-D532N-L223A-K224A-E227A, GFP-PKCε-D532N-T228A or GFP-PKCε-D532N-S234A accumulation at the midbody. Values plotted are means for n = 3 experiments, with error bars denoting SD. Thirty cells with midbodies were observed per condition per experiment. One-way analysis of variance: **** = P ≤ 0.0001; *** = P ≤ 0.001; ns (not significant) = P > 0.05. (D) Representative immunofluorescence images of HEK293T cells expressing GFP-PKCε, GFP-PKCε-D532N or GFP-PKCε-L223A-K224A-E227A and treated with DMSO, PMA or diC10 as indicated. Cells were treated for 20 min with 1 µM PMA, or 3 min with 10 µM diC10. Cells stained for DNA (blue) and tubulin (red), with GFP-PKCε shown in grey; the midbody region highlighted is expanded for each image. Twenty cells with midbodies were observed per condition across three experiments. Scale bar = 20 µm.