Figure 4

Effects of PKCι depletion on Lgl. A. U87MG cells expressing either empty vector (-) or vector expressing Flag-tagged Lgl (+) were either mock-transfected (-) or transfected with an RNA duplex A targeting PKCι (+). Three days after transfection, cells were lysed and Flag-tagged Lgl was immunoprecipitated. Immunoprecipitates were solubilized in Laemmli buffer, electrophoresed on SDS-PAGE gels and transferred to membranes for analysis by Western blotting. Membranes were probed with antibodies to Flag epitope or phosphorylated PKC substrate, as indicated. In the lane marked "nl", the immunoprecipitation was performed with no lysate (buffer only). B. U87MG/Lgl cells were grown on gelatin-coated coverslips and transfected with either scrambled duplex, RNA duplex A targeting PKCι or RNA duplex C targeting PKCι. Two days later cells were fixed and immunocytochemistry for Flag-tagged Lgl (red) and non-muscle myosin IIA (green) was performed. Nuclei were stained with DAPI.