Figure 8. KD of -actinin-4 in MDCK cells by shRNA does not alter the overall epithelial morphology but disrupts actin assembly at the apical junction. (a) Western blot analysis of -actinin-4 in parental MDCK and clonal cell lines of two different shRNAs against -actinin-4. Each lane contains an equal amount of total protein. (b) Single deconvolved optical z slices at the apical region of cells showing -actinin-4 (red) expression correlating with levels of apical actin (phalloidin; blue). (c) Single deconvolved optical z slices at the apical region of latrunculin B–treated cells showing -actinin-4 levels not correlating with latrunculin-stable actin puncta. (d) Single deconvolved optical z slices at the apical region of cells showing -actinin-4 expression correlat- ing with actin assembly after latrunculin washout. (e) Projections of 11 deconvolved optical z slices spanning the apical 2 µm of a mixed population of -actinin-4 KD cells showing normal levels of -catenin (green). White arrowheads point to junctions with diminished actin between cells with -actinin-4 KD. (f) Projections of 11 deconvolved optical z slices spanning the apical 2 µm of a mixed population of -actinin-4 KD cells treated with latrunculin B. Formation of latrunculin-stable actin puncta is unaffected by -actinin-4 KD. (e and f) Yellow arrowheads point to apical actin puncta formed between cells 124 JCB • VOLUME 196 • NUMBER 1 • 2012