Fig. 1. Knockdown of mLgl retains apical proteins and F-actin at cell periphery in depolarized MDCK cells. (A) Western blot analysis of control cells (21-1 and 21-7) and mLgl-1-mLgl-2 double-kd MDCK cell clones (24-15 and 24-16) with antibodies against indicated proteins. mLgl-1 and mLgl-2 expression levels were specifically reduced in clones 24-15 and 24-16. The mLgl knockdown retains the apical proteins and F-actin at the cell periphery in depolarized MDCK cells. (B) Confluent monolayer of control cells or mLgl kd cells on permeable filter were incubated with LC medium for 20 hours, after which cells were fixed and co-stained with anti-gp135 antibody and Rhodamine-phalloidin. Arrowheads indicate cells with intracellular gp135 and F-actin staining. (C) Cells treated as in B, stained with antibodies against ezrin and ZO-1. Arrowheads indicate cells with intracellular ezrin staining. (D) Quantification of the cells with peripheral gp135 staining. The percentage of total cells is shown. Values are mean (±s.d.) of three independent experiments (21-1 and 24-15) or mean of two independent experiments (24-16). Each photograph represents the projected views of optical sections from the apical to the basal membranes of cells. Bars, 20 µm.