Figure 1. Deletion of Srd5a3 in the cerebellum impairs protein N-glycosylation and motor function.
(A) Far-WB with SNA lectin in P7 cerebellum and (B) quantification (n = 4 per genotype), p-value=0.29 (C) WB analysis of LAMP1 expression in mouse embryonic fibroblasts (MEFs). (D) WB analysis of LAMP1 level from P7 cerebellum and quantification (E). * indicates a PNGase sensitive LAMP1 isoform; **p-value=0.0032. (F) WB analysis as described in (D) but with increased electrophoretic migration and adjusted protein amounts to highlight LAMP1 hypoglycosylation in the mutant sample. (G) Box plot of the latency to fall during rotarod testing (n = 15 for each condition). One-way ANOVA was used for rotarod statistics. For all others, two-tailed student t-test was used. **p<0.01; ***p<0.001. Results are presented as mean ±s.d.
Figure 1—figure supplement 1. Generation of conditional Srd5a3 knockout mice and extended biochemical and behavioral analyses.
(A) Schematic of the Srd5a3 knockout-first allele (tm1a), the same allele after Flp-mediated recombination (tm1c) and (B) validation of the tm1a allele insertion by Southern blot. (C) RT-qPCR analysis of Srd5a3 expression levels in the P7 cerebellum (Control, n = 3; En1-Cre; Srd5a3fl/-, n = 4) (D) RT-PCR analysis of the recombined (KO) and wild-type (WT) Srd5a3 transcripts in the cerebellum. (E) LacZ staining image of the En1Cre; Rosa26 mouse showing Cre expression within the entire cerebellum. Scale bar 2 mm. (F) ConA far-WB and (G) quantification (t-test, p-value=0.7190; n = 3 for each genotype). (H) Average swimming speed during the Morris water maze test (MWM; n = 15 for each genotype). (I) MWM results. After two trials to find the same hidden platform (average of a total of eight sessions), only control mice significantly reduced the time to find the platform on the second trial (t-test, type I, p=0.0329). Unless indicated, two-tailed Student t-test was used for statistics. *p<0.05; ***p<0.001.