PM localization of PKCζ in HEK293 cells requires both polybasic PSr and Par-6. (A) PKCζ::GFP or Par-6::RFP was cytosolic when expressed alone, but both became strongly PM-localized when coexpressed. PKCζ::RFP^-2A and ^2A-Par-6::iRFP also showed strong PM localization. (B) Pan-aPKC antibody (anti-aPKC) detects both exogenously expressed PKC_L::GFP and PKCζ::GFP in HEK293 cells (white arrowhead), as well as endogenous expressed aPKC (black arrowhead). PKCζ-specific antibody (anti-PKCζ) specifically detected exogenously expressed PKCζ::GFP but showed no detectable expression of endogenous PKCζ in HEK293. α-Tubulin serves as loading control. (C) Only PKCζ::RFP^-2A (∼100 kD) but not full-length PKCζ::RFP^-2A-Par-6::iRFP fusion protein (∼150 kD) was detected in cells expressing PKCζ::RFP^-2A-Par-6::iRFP. Lysate from cells expressing PKCζ::RFP was loaded and blotted as a positive control. (D) PKCζ^C107Y::GFP (C107Y), PKCζ^KR8Q::GFP (KR8Q), and PKCζ^ΔPSr::GFP (ΔPSr) did not localize to PM when coexpressed with Par-6::RFP. (E) Both PKCζ::GFP and PKCζ^KR8A::GFP, but not PKCζ^C107Y::GFP, coimmunoprecipitated with FLAG-Par-6 by anti-GFP antibody from HEK293 cells. In all data plots, boxes extend from 25th and 75th percentiles, with lines in the middle indicating the median and whiskers indicating 10th and 90th percentiles. Sample numbers are indicated in parentheses at the right. Orange dashed lines in quantification figures indicate that the PM localization index = 1 (see Materials and methods). Measurements <1 indicate cytosolic localization, and those >1 indicate PM localization. PM localization axes in all figures are in log2 scale. Scale bars: 5 µm (A and D).