Fig. 3. ABBA is abundantly expressed in glial cell lines where it localises to the interface between plasma membrane and cortical actin cytoskeleton. (A) Western blot analysis demonstrating abundant expression of ABBA in primary glia, whereas cortical neurons derived from E16.5 mouse embryos are negative for ABBA. Anti-actin was used a loading control. (B) ABBA is expressed in glial cell lines (C6 and C6-R), but was not detected in neuronal cell lines (N18, Neuro2A, or Shep). As a loading control, anti-actin antibody was used. (C-E) Immunofluorescence microscopy images from C6-R cells revealed that endogenous ABBA localises in lamellipodia at the leading edge of the cortical actin cytoskeleton (white arrowhead). (F-H) Co-labelling of C6-R cells with ABBA antibody and membrane marker (CM-Dil) demonstrates that ABBA localises to the plasma membrane at the leading edge. (H) Magnified areas show individual channels from the boxed region of the image. (I-K) Three-dimensional confocal microscopy analysis from C6-R cells confirmed the localisation of ABBA to the interface between plasma membrane and the actin cytoskeleton (white arrowheads). ABBA is in green whereas F-actin (panels D,E,I,J,K) and plasma membrane (panels G,H) are in red. Scale bars: 20 µm in C-H; 5 µm in I; 2 µm in J,K.