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. 2015 Aug 19;35(33):11514–11531. doi: 10.1523/JNEUROSCI.5288-14.2015

Figure 1.

Figure 1.

Generation of transgenic mice expressing a fusion protein, either UbG76V-GFP-Syb2 or GFP-Syb2. A, Schematic diagrams of constructs for generating transgenic mice expressing either UbG76V-GFP-Syb2 or GFP-Syb2, directed by a neuron-specific Thy1 promoter. The asterisk (ub*) denotes a point mutation (G76V) at the last residue of the C terminus of the Ub, which prevents the removal of Ub from its substrate. B, Western blot analysis of total brain and spinal cord homogenates prepared from 3-month-old nontransgenic control, GFP-Syb2, and UbG76V-GFP-Syb2 mice, probed with anti-GFP antibodies and then reprobed with antibodies against VCP. Anti-GFP antibodies recognize a single band predicted by the size of the fusion protein, either GFP-Syb2 or UbG76V-GFP-Syb2. C, D, Western blot analysis of spinal cord homogenates prepared from nontransgenic (control), UbG76V-GFP-Syb2 transgenic (line 1), and UbG76V-GFP-Syb2 transgenic (line 2) mice (three mice in each group, 3 months of age) and probed with antibodies against Syb2, which recognizes both endogenous Syb2 and UbG76V-GFP-Syb2. The blot was reprobed with anti-syntaxin1 antibodies. The endogenous Syb2 was abundant in both nontransgenic and transgenic mice, whereas UbG76V-GFP-Syb2 was detected only in the transgenic mice at a level markedly lower than those of the endogenous Syb2. This is quantified by the bar graph on the right comparing the levels of UbG76V-GFP-Syb2 expression relative to the endogenous Syb2 (D). E, F, Western blot analysis of spinal cord homogenates from nontransgenic (control), GFP-Syb2 transgenic, and UbG76V-GFP-Syb2 transgenic (line 1) mice (three mice in each group, 3 months of age), probed with antibodies against Syb2. Relative expression levels (compared with endogenous Syb2) are shown in the bar graph (F). G, H, Western blot analysis of spinal cord homogenates from nontransgenic control and UbG76V-GFP-Syb2 transgenic mice (3, 6, and 8 months of age, three pairs of mice in each age group). Expression levels of UbG76V-GFP-Syb2 remained at a steady-state level at 3, 6, and 8 months of age (H).