Additional file 1 of Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain
Creators: name: Singh, Vijay
name: Menard, Marissa A.
name: Serrano, Geidy E.
name: Beach, Thomas G.
name: Zhao, Hien T.
name: Riley-DiPaolo, Alexis
name: Subrahmanian, Nitya
name: LaVoie, Matthew J.
name: Volpicelli-Daley, Laura A.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology

description: Additional file 1. Intracerebroventricular injected C57Bl/6 WT mice with control ASO and Rab10 ASOs, ASO1 and ASO2, and G2019S-LRRK2 KI mice to confirm the specificity of the Rab10 and pRab10 antibodies. At 3–4 months of age, C57BL6/J WT mice received intraventricular injections with control and Rab10 specific ASOs. Mice were deeply anesthetized with vaporized isoflurane on a stereotactic frame. Mice were then injected with 10 µL of 30 µg/µL (total 300 µg) control and Rab10 ASOs using the coordinates + 0.3 mm AP, + 1.0 mm ML, -3.0 mm DV. Solutions were injected at a constant rate of 1 µL/min; once injection was complete, the needle was left in place for 5 min and then slowly withdrawn (a) Immunoblot of Rab10 in Rab10 ASOs (Rab10 ASO1 and Rab10 ASO2) and control ASO injected brain samples for cortex, midbrain and striatum. Tubulin was used as a loading control. (b) Quantitation of Rab10 protein and normalization with the loading control, tubulin, show reduction of the Rab10 in the Rab10 ASO1 compared to control ASO injected mouse samples in the cortex (p value = 0.0126), midbrain (p value = 0.0239) and in the striatum (p value = 0.0235) and reduction of the Rab10 in the Rab10 ASO2 compared to control ASO injected mouse samples in the cortex (p value = 0.0044), midbrain (p value = 0.0208) and in the striatum (p value = 0. 0133). One way ANOVA Dunnett’s multiple comparisons test was run for statistical analysis (n = 3 biologically independent samples). (c) Immunoblot of pRab10 in Rab10 ASOs (Rab10 ASO1 and Rab10 ASO2) and control ASO injected brain samples for cortex, midbrain and striatum. Tubulin was used as a loading control. (d) Quantitation of pRab10 protein and normalization with the loading control, tubulin, show reduction of the pRab10 in the Rab10 ASO1 compared to control ASO injected mouse samples in the cortex (p value = 0.0594), midbrain (p value = 0.0205) and in the striatum (p value = 0.1705) and reduction of the pRab10 in the Rab10 ASO2 compared to control ASO injected mouse samples in the cortex (p value = 0. 0544), midbrain (p value = 0.0192) and in the striatum (p value = 0.1087). One way ANOVA Dunnett’s multiple comparisons test was run for statistical analysis (n = 3 biologically independent samples). (e) Immunofluorescence for pRab10 (green) in the WT and G2019S KI mouse striatum region showed an increase of pRab10 in G2019S KI brain samples. NeuN (red) staining was used to show comparable immunofluorescence in the brain area. Scale bar = 500 µm. All images were captured at the same laser power, gain and offset. (f) The zoomed in image showed increased pRab10 (green) immunofluorescence in G2019S KI samples. Scale bar = 200 µm. All images were captured at the same laser power, gain and offset. (g) Quantitation of pRab10 integrated density immunofluorescence signal and normalization with NeuN signal showed a significant increase (p value = 0.004) in the G2019S KI samples (gray bar) compared to WT samples (black bar) (n = 3 biologically independent samples). For statistical analysis two tailed nested t-test was performed.