Journal: Communications biology
Article Title: RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.
doi: 10.1038/s42003-023-04826-x
Figure Lengend Snippet: Fig. 5 Quantitative proteomics in Rab35 cKO P0 hippocampus. a Volcano plot of the TMT-based quantitative proteomes identifying the dysregulated proteins in Rab35 cKO hippocampus in comparison with the control hippocampus (n = 5 mice per genotype). b Number of proteins identified as significantly dysregulated and as either membrane traffic-related or neuronal migration-related. c, d Western blot analysis of control and Rab35 cKO P0 hippocampi using anti-contactin-2, anti-CHL1, and anti-actin antibodies. e, f Quantification of contactin-2 (c) and CHL1 (d) protein levels in control and Rab35 cKO P0 hippocampi. Band intensities of the indicated proteins were normalized to those of actin (n = 9 mice per genotype). Unpaired Student’s t- test; e, p = 0.0153; f, p = 0.0095. g, h Levels of contactin-2 (g) and CHL1 (h) were quantified by targeted MS using the PRM method (n = 5 mice per genotype). Unpaired Student’s t-test; g p = 0.0053; h p = 0.0229. i Western blot analysis of control and Rab35 cKO P0 hippocampus using anti-N-cadherin and anti-actin antibodies. j Quantification of N-cadherin protein levels in the control and Rab35 cKO P0 hippocampus (n = 9 mice per genotypes). Unpaired Student’s t-test, p = 0.9020. k Representative images of DIV 2 hippocampal primary neurons stained for contactin-2 (green), rhodamine-phalloidin (magenta) and DAPI (blue). Scale bar, 20 μm. l Quantification of contactin-2 intensity at the somatic plasma membrane in control (n = 4) and Rab35- deficient (n = 4) cells. Thirty neurons from four different cultures per genotype were analyzed. Mann–Whitney U-test, p = 0.0286. Data represent the mean ± SEM; n.s. not significant (p > 0.05); *p < 0.05; **p < 0.01.
Article Snippet: The following primary antibodies were used for immunoblotting: 1:1000 RAB35 (rabbit; Cell Signaling, 9690 S), 1:10,000 Actin [C4] (mouse; MerckMillipore, MAB1501), 1:10,000 GAPDH [6C5] (mouse; Merck-Millipore, MAB374), 1:1000 contactin-2/TAG-1 (goat; R&D systems, AF4439), 1:1000 CHL1 (goat; R&D systems, AF2147), and 1:1000 N-cadherin [32/N-cadherin] (rabbit; BD, 610920).
Techniques: Quantitative Proteomics, Comparison, Control, Membrane, Migration, Western Blot, Staining, Clinical Proteomics, MANN-WHITNEY
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