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Figure 1 | Acta Neuropathologica Communications

Figure 1

From: Amyotrophic lateral sclerosis (ALS)-associated VAPB-P56S inclusions represent an ER quality control compartment

Figure 1

Generation of VAPB-WT and VAPB-P56S transgenic mice. A) To generate VAPB transgenic mice the cDNAs of wild-type or P56S-mutant human VAPB coupled to HA were cloned into the Thy1.2-expression casette. B-E) Western blots showing relative VAPB transgene expression levels in tissues of Thy1.2-hVAPB-WT (VW2, VW3) and Thy1.2-hVAPB-P56S (VM1, VM2, VM3) mice. Transgenic VAPB is detected with anti-HA antibody that specifically detects the transgene (B) or anti-VAPB antibody that interacts with both endogenous VAPB and transgenic VAPB running in a higher molecular weight band because of the HA-tag (B, D, E). Each lane is loaded with 2.5 μl S1 fraction derived from 250 μg tissue. B, C) Representative results (B) and quantification (C) of Western blot of spinal cord homogenates showing relatively high transgene expression levels in wild-type VAPB expressing lines (VW2 and VW3), and moderate transgene expression in mutant VAPB lines (VM1, VM2). Values in C are expressed as the ratio of the signals of endogenous and transgenic VAPB and represent means ± SE (n > 3). Spinal cords from VM2 mice show about half the level of transgene expression compared to VM1 spinal cord (Student t-test). D) Western blot of homogenates from different tissues from VM1 and VM3 mice showing that the transgene is specifically expressed in nervous system. E) Western blot of sciatic nerve homogenate of wild-type (line VW3) and mutant (line VM1) transgenic mice showing a high level of transgenic VAPB in wild-type VAPB sciatic nerve, and no transgenic VAPB in mutant VAPB sciatic nerve. F) Anti-HA immunohistochemistry showing widespread transgene expression in the brain of wild-type and mutant VAPB transgenic mice.

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