Figure 3

VAPB determines surface expression and dendritic localization of HCN2. A) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope (^EGFPHCN2^HA_Ex) alone or cotransfected with VAPB or the TM segment of VAPB (TM^VAPB). B) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for ^EGFPHCN2^HA_Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 protein expression. C) Chemiluminescence surface expression assay as in B, but using TM^VAPB (1.6 ± 0.1). D) Immunocytochemistry of ^HAVAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with ^HAVAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E) Immunocytochemistry experiment as in D, but transfecting the ALS8 mutation ^HAVAPB^P56S (magenta), leading to an aggregation of VAPB^P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm (A, D, E). All data are presented as means ± sem. The number of experiments (n) is indicated in the respective bar graphs. **P < 0.01 (unpaired Student’s t test).