VAPB Inclusions in VAPB-P56S transgenic motor neurons are immunoreactive for ER markers and surrounded by ribosomal markers. A, B) Confocal immunofluorescence of HA (specific for transgenic VABP) and VAPB (labels endogenous and transgenic VAPB) in motor neurons of non-transgenic (A) and mutant VAPB (line VM1; B) mice showing multiple small intensely HA and VAPB-immunoreactive inclusions in VM1 motor neurons. Note, that VAPB-immunoreactivity in the rest of the cell body is the same as in non-transgenic mice. C-F) Confocal immunofluorescence of spinal motor neurons double-labeled with either anti-HA; C, D) or anti-VAPB; E, F) and antibodies against ER proteins, i.e. calreticulin (C, D) or KDEL-motif proteins (E, F); calreticulin and KDEL-immunoreactivities are diffusely distributed throughout the perykarya of both non-transgenic (C, E) and mutant VAPB (D’, F’) motor neurons irrespective of the presence of VAPB inclusions, but G, H) Double labeling for HA and the ribosomal proteins P0 (G) or phosphorylated-S6 (H) shows that mutant VAPB inclusions are P0 and phospho-S6-immunonegative but the surrounding cytoplasm is always intensely P0 (arrows in G) and phospho S6-positive (H). Arrow and insert in H show that also dendritic VAPB inclusions are surrounded by high levels of ribosomes. I-L) Double labeling of VAPB or HA, with antibodies against the cis-Golgi protein GM130 (I, J), lysosomal protein LAMP1 (K), and the FFAT-motif protein ORP9 (L), shows that VAPB inclusions (arrows in K and L) are immunonegative for these proteins. Note the presence of autofluorescent structures, representing lipofuscin (aging pigment) in the motor neuron shown in L, which is from a 70 week old VM1 mouse. Bar in A, 10 μm.