PM-targeted PKCζ/Par-6 complex is inhibited from phosphorylating Lgl. (A) PM localization of Lgl::GFP was strongly reduced in cells expressing PKCζ::RFP but not kinase-dead PKCζ^K281W::RFP. Nonphosphorylatable Lgl^S6A::GFP remained PM-localized in cells expressing PKCζ::RFP. Lgl::GFP remained PM-localized in cells coexpressing PKCζ::RFP and Par-6::iRFP. Lgl::GFP also showed strong PM localization in cells expressing PKCζ::RFP^-2A-Par-6::iRFP. (B) PM localization of Lgl::GFP was strongly reduced in cells expressing Lyn11-PKCζ::RFP, PKCζ^KR8Q::RFP (KR8Q), and Lyn11-PKCζ^KR8Q::RFP (Lyn11-KR8Q). In all three cases, coexpression of Par-6::iRFP increased PM localization of Lgl::GFP. (C) Cells expressing Lgl::GFP only, expressing both Lgl::GFP and PKCζ::RFP (or PKCζ^KR8Q::RFP or Lyn11-PKCζ^KR8Q::RFP), or expressing Lgl::GFP together with PKCζ::RFP (or PKCζ^KR8Q::RFP or Lyn11-PKCζ^KR8Q::RFP) and FLAG::Par-6, were directly lysed in SDS loading buffer and analyzed by Western blot. Anti-(P)-Lgl, antibody against phosphorylated Lgl. All experiments were performed in HEK293 cells. Scale bars: 5 µm.