Journal: Cell reports
Article Title: A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum.
doi: 10.1016/j.celrep.2020.107703
Figure Lengend Snippet: Figure 5. Sam68 Preferentially Represses Exons Characterized by a Weak Branchpoint 5368289352488500(A and B) Branchpoint score (A) and GC content (B) for up- and downregulated exons in Sam68KO cerebella with respect to non-regulated exons (Wilcoxon rank-sum test). (C) Distribution of the DPSI of exons differentially regulated between Sam68KO and WT cerebella. Whiskers indicate 1.5 interquartile range. Welch’s t test; ****p < 0.0001. (D and E) Left panels: scheme of the AS region of the Arhgef9 (D) and Arhgap12 (E) gene, with regulated (red) and constitutive exons (green) and qPCR primers (arrows) and the splicing events in WT and Sam68KO cerebella. Shown is RT-PCR analysis for Sam68-regulated events in Arhgef9 (D) and Arhgap12 (E) with PSI values from densitometric analysis (mean ± SEM, n = 6, paired t test). Right panels: bar graphs showing qPCR analyses of Sam68 CLIPs for Arhgef9 (D) and Arhgap12 (E) pre-mRNAs in P10 mouse cerebella. Immunoglobulin G (IgG) was used as a control (mean ± SEM, n = 5, two tailed paired t test, *p < 0.05). (F and G) qPCR analyses of U2AF65 CLIPs for Arhgef9 (F) and Arhgap12 (G) pre-mRNAs in WT and Sam68KO P10 cerebella (mean ± SEM, n = 5, multiple Student’s t test, *p < 0.05). (H) Western blot analysis of U2AF65 and Sam68 expression in WT and Sam68KO P10 mouse cerebella. See also Figure S5.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-Sam68 Bethyl Laboratories Cat#A302-110A; RRID:AB_1604289 Donkey whole antibody anti Rabbit IgG HRP-linked GE Healthcare Cat# NA934; RRID: AB_772206 Sheepwhole antibody anti mouse IgGHRPlinked GE Healthcare Cat# NA931; RRID:AB_772210 Alexa Fluor-488 goat anti mouse Thermo Fisher Scientific Cat# A11001; RRID:AB_2534069 Alexa Fluor-594 goat anti-rabbit Thermo Fisher Scientific Cat# A11037; RRID:AB_2534095 Mouse monoclonal anti 5-bromo-20deoxyuridine (BrdU) Millipore Cat# 05-633, RRID:AB_11212826 Mouse monoclonal anti CALB1 Sigma-Aldrich Cat# C9848, RRID:AB_476894 Mouse monoclonal anti-U2AF65 Sigma-Aldrich Cat#U4758, RRID:AB_262122 Rabbit polyclonal anti VGLUT1 Synaptic System Cat# 135 303, RRID:AB_887875 Mouse monoclonal anti-ACTIN Santa Cruz Cat# sc-47778, RRID: AB_626632 Chemicals, Peptides, and Recombinant Proteins Dnase I from bovine pancreas Sigma Aldrich Cat# DN25; CAS 9003-98-9 Trypsin from bovine pancreas Sigma Aldrich Cat#T8003 Poly-L-lysine Sigma Aldrich Cat# P9155 Trypsin inhibitor from bovine pancreas Sigma Aldrich Cat#T9003 5-Bromo-20-deoxyuridine Sigma Aldrich Cat#B5002 B27tm Supplement Thermo Fisher Scientific,GIBCO Cat#17504-044 (+)-Bicuculline Tocris Cat#0130 Tetrodotoxin Tocris Cat#1078 RNAprotect Tissue Reagent QIAGEN Cat# 76104 Tissue-Tek O.C.T.
Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test, Western Blot, Expressing
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