The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization
Fig 3
MARCKS mutant expression and localization in U87 glioma cells.
A) Diagram depicting the domains of the engineered MARCKS wild-type (WT) and effector domain deleted (ΔED) lentiviral constructs. Starting at the N-terminus there is a myristoylation domain (N-MYR), MH2 domain (N-MH2), an effector domain (WT-ED) with amino acid sequence indicated, and lastly a C-terminus V-5 tag. B) Western blot showing doxycycline inducible MARCKS mutant over-expression in U87 cells. Nuclear and cytoplasmic fractions were prepared and separated by SDS-PAGE and probed for V-5 (for MARCKS expression), lamin and tubulin for nuclear and cytoplasmic fraction, respectively. C) Confocal microscopy is shown for U87 WT-MARCKS (WT) and ΔED-MARCKS (ΔED) cells were fixed and stained for MARCKS (V5), nucleus (DAPI), and PIP2 with the merged image shown on the right. D) High magnification of the PIP2 staining of the nucleus with punctate staining marked with red arrows.
