Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Normalized counts of the three RBFOX genes in striatal RNA from 3.5 month-old WT (n=3) and R6/1 mice (n=3) according to RNA-seq datasets in Elorza et al . 15 ( B ) Quantification of Rbfox1 transcript levels by RT-qPCR in striatal RNA from 3.5 month-old WT (n=7) and R6/1 mice (n=7) (Student’s t-test; * P < 0.05). ( C ) Rbfox1 immunohistochemistry in striatum of 3.5 month-old WT and R6/1 mice. ( E ) Representative RBFOX1 immunohistochemistry staining in striatum of control and HD subjects.

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Immunohistochemistry, Staining, Control

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Mice expressing tTA under control of the CamKII promoter (CamKII-tTA mice) were bred with mice carrying the β-Gal-BiTetO-RBFOX1 or β-Gal-BiTetO-U2AF2 construct to yield TgRBFOX1 (CamKII-tTA:β-Gal-BiTetO-RBFOX1) mice or TgU2AF2 (CamKII-tTA:β-Gal-BiTetO-U2AF2) . ( B ) Immunohistochemistry with anti-RBFOX1 or anti-U2AF2 antibody in sagittal sections from 1.5 month-old WT, TgRBFOX1 or TgU2AF2 mice.

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: Expressing, Control, Construct, Immunohistochemistry

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: (A) Gel shows RT-PCR amplification of Tg-RBFOX1 or TgU2AF2 mRNA in wild-type, MildTgRBFOX1 or MildTgU2AF2 and StrongTgRBFOX1 or StrongTgU2AF2 mice. (B) Histogram shows brain weight of WT (n=5/n=8), MildTgRBFOX1 (n=7) or MildTgU2AF2 (n=5) and Strong TgRBFOX1 (n=7) or Strong TgU2AF2 (n=6) mice (ANOVA, followed by Tukey’s post hoc test; *P < 0.05;**P < 0.01; ***P < 0.001).

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: bioRxiv

Article Title: Correction of RBFOX1 deficit rescues Huntington’s disease mis-splicing and pathology

doi: 10.1101/2024.11.06.622223

Figure Lengend Snippet: ( A ) Venn diagram showing the 83 genes in the intersection between the 245 genes with exons mis-spliced in Huntington’s disease 15 and the 543 genes with RBFOX-direct target exons 25 ( B ) Venn diagram showing the 76 genes in the intersection between the 245 genes with exons mis-spliced in Huntington’s disease 15 and the 966 genes that are functional targets of Rbfox1 according to Supplementary Table 1. Representation factor (RF) was determined with Two-sided Fisher’s Exact test, using as background genes the human-mouse orthologous genes coincidentally detected in the human and mouse RNA-seq datasets used to define the Huntington’s disease mis-splicing signature 15 ( n = 12,882).

Article Snippet: Antibodies: rabbit anti-RBFOX1 (1:5000, Novus, NBP1-90304), rabbit anti-β-Gal (1:2000, Invitrogen, A-11132), rabbit DARPP32 (1:3000, BD, 611520) and rabbit Cleaved caspase-3 (1:100, Cell Signaling, 9661) for mouse samples.

Techniques: Functional Assay, RNA Sequencing Assay

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: Primer sequences.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: Quantitative RT-PCR, Clone Assay

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: Prediction of splicing regulator RNA binding proteins (RBPs) for each AS event by rMAPS2 in the DRGs of mice with SNL vs. sham surgery. (a) Summary of the RBPs motif maps with the most significant enrichment for each up- and downregulated AS event. (b) Relative mRNA levels of top three enriched RBPs for up- and down-regulated SE events in the ipsilateral L 4 DRGs of mice after SNL or sham surgery (n ​= ​3 per group). (c) RNA motif maps showing enrichment of Rbfox1 near exons that are alternatively spliced. Maps for Rbfox1 motif enrichment for SE events from the rMATS analysis of SNL mice DRGs are shown for silenced (red) and enhanced (blue) splicing events. The dotted lines indicate the significance of enrichment versus background. The solid lines indicate the motif score of upregulated and downregulated genes compared to background genes.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: RNA Binding Assay

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: Peripheral nerve injury induced decreased Rbfox1 mRNA and protein in the injured DRGs. (a) Rbfox1 mRNA expression in the ipsilateral L 4 DRG of mice after SNL or sham surgery. n ​= ​12 mice/time point/group. ∗∗ P ​< ​0.01, according to two-way ANOVA followed by a post hoc Tukey's test. (b) Rbfox1 protein expression in the ipsilateral L 4 DRG of mice after SNL or sham surgery. Representative Western blots (left panels) and a summary of densitometric analysis (right graphs) are shown. n ​= ​12 mice/time point/group. ∗ P ​< ​0.05, ∗∗ P ​< ​0.01, according to two-way ANOVA followed by a post hoc Tukey's test. (c) Rbfox1 protein expression in the contralateral L4 DRG, ipsilateral L3 (intact) DRG, and ipsilateral L4 spinal cord of mice after SNL. Representative Western blots (left panels) and a summary of densitometric analysis (right graphs) are shown. n ​= ​12 mice/time point/group. (d) Rbfox1 mRNA expression in the ipsilateral L 3/4 DRGs of mice on day 7 after CCI or sham surgery. n ​= ​6 mice/group. ∗∗ P ​< ​0.01, according to a two-tailed, independent-sample Student's t -test. (e) Rbfox1 protein expression in the ipsilateral L 3/4 DRGs of mice on day 7 after CCI or sham surgery. n ​= ​6 mice/group. ∗∗ P ​< ​0.01, according to a two-tailed, independent-sample Student's t -test. (f) Rbfox2 protein expression in the ipsilateral L 4 DRG of mice after SNL or sham surgery. n ​= ​12 mice/time point/group. ∗∗ P ​< ​0.01, according to two-way ANOVA followed by a post hoc Tukey's test. Representative Western blots (upper panels) and a summary of densitometric analysis (below graphs) are shown.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: Distribution pattern of Rbfox1 protein in the DRG of naive mice and changes in the number of Rbfox1-positive neurons in injured DRGs after SNL. (a, b) Representative photographs show that Rbfox1 (red color) is coexpressed exclusively with NeuN (a) and undetectable in glutamine synthetase (GS)-labeled cells (b). Cellular nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). n ​= ​3 mice. Scale bars: 200 ​μm for a and b. (c) Histogram displaying the distribution of Rbfox1-positive neuronal somata in DRGs. Small: 58 ​%. Medium: 27 ​%. Large: 17 ​%. (d) Changes in the number of Rbfox1-positive neurons in the ipsilateral L 4 DRG on day 7 after SNL or sham surgery. n ​= ​3 mice/group. ∗∗ P ​< ​0.01, according to a two-tailed, independent-sample Student's t -test. Scale bar: 100 ​μm.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: Labeling, Two Tailed Test

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: DAPI-stained images of the Rbfox1 protein and several neuronal markers in the DRGs. (a) Rbfox1-positive neurons were labeled by isolectin B4 (IB4), (b) Venn diagram shows the number of neurons double stained by Rbfox1 protein and IB4. (c) Rbfox1-positive neurons were labeled by calcitonin gene-related peptide (CGRP), (d) Venn diagram shows the number of neurons double stained by Rbfox1 protein and CGRP. (e) Rbfox1-positive neurons were labeled by neurofilament-200 (NF200) in naive DRG (n ​= ​3 mice). Scale bars: 200 ​μm. (f) Venn diagram shows the number of neurons double stained by Rbfox1 protein and NF200. n ​= ​3 mice.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: Staining, Labeling

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: Rescuing diminished DRG Rbfox1 inhibits the development of pain hypersensitivity and spinal cord dorsal horn central sensitization in male mice induced by SNL. (a, b) Rbfox1 and Rbfox2 protein expression in the ipsilateral L 4 DRG (a) and the level of Rbfox1 protein in the ipsilateral L 3 DRG and ipsilateral L 4 spinal cord (b) on day 14 following SNL or sham surgery in male mice microinjected with AAV5- Rbfox1 or AAV5- Gfp . n ​= ​12 mice/group. ∗ P ​< ​0.05 vs . sham male mice microinjected with AAV5- Gfp at the corresponding time point. # P ​< ​0.05 vs . SNL male mice microinjected with AAV5- Gfp at the corresponding time point. One-way ANOVA followed by a post hoc Tukey's test. (c–i) Paw withdrawal frequency in response to low (0.07 ​g; c, g) and median (0.4 ​g; d, h) force von Frey filament stimuli and paw withdrawal latency in response to heat (e, i) and cold (f) stimuli on the ipsilateral side (c–f) and contralateral side (g–i) of male mice with microinjection of AAV5- Rbfox1 or AAV5- Gfp into the ipsilateral L4 DRG at different days following SNL or sham surgery. n ​= ​12 mice/group. ∗∗ P ​< ​0.01 vs . sham male mice microinjected with AAV5- Gfp at the corresponding time point. # P ​< ​0.05, ## P ​< ​0.01 vs . SNL male mice microinjected with AAV5- Gfp at the corresponding time point. Two-way repeated measures ANOVA followed by a post hoc Tukey's test. (I) The levels of phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, and GFAP in the ipsilateral L 4 spinal cord dorsal horn of male mice on day 14 after SNL or sham surgery. n ​= ​3 mice/group. ∗∗ P ​< ​0.01, according to two-way ANOVA followed by Tukey's post hoc test.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: Expressing

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: Locomotor function.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: shRNA

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: Rescuing the reduction in DRG Rbfox1 caused by SNL mitigated the maintenance of SNL-induced pain hypersensitivity and spinal cord dorsal horn central sensitization in male mice. Male mice were subjected to SNL 14 days following DRG microinjection of AAV5- Rbfox1 or AAV5- Gfp . (a–g) Paw withdrawal frequency in response to low (0.07 ​g; a, e) and median (0.4 ​g; b, f) force von Frey filament stimuli and paw withdrawal latency in response to heat (c, g) and cold (d) stimuli on both the ipsilateral (a–d) and contralateral (e–g) sides of male mice with microinjection of AAV5- Rbfox1 or AAV5- Gfp into the ipsilateral L 4 DRG at different days after SNL. N ​= ​12 mice/group. ∗ P ​< ​0.05, ∗∗ P ​< ​0.01 vs. SNL male mice microinjected with AAV5- Gfp at the corresponding time point on the ipsilateral side. Two-way repeated measures ANOVA followed by a post hoc Tukey test. (h) Rbfox1 protein in the ipsilateral L 4 DRG of naive and AAV5- Rbfox1- or AAV5- Gfp -microinjected male mice on day 28 following SNL or sham surgery. n ​= ​12 mice/group. ∗ P ​< ​0.05 vs. naive male mice. # P ​< ​0.05 vs. SNL male mice microinjected with AAV5- Gfp . One-way ANOVA followed by Tukey's post hoc test. (I) Total ERK1/2, p-ERK1/2 and GFAP levels in the ipsilateral L 4 spinal cord dorsal horn on day 28 following SNL or sham surgery in male mice with DRG microinjection of AAV5- Rbfox1 or AAV5- Gfp . n ​= ​3 mice/group. ∗ P ​< ​0.05, ∗∗ P ​< ​0.01 vs. naive male mice. # P ​< ​0.05 vs. SNL male mice microinjected with AAV5- Gfp . One-way ANOVA followed by Tukey's post hoc test.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques:

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: DRG Rbfox1 knockdown produced an enhanced pain response and spinal cord dorsal horn central sensitization in naive male mice. (a) The level of Rbfox1 in the ipsilateral L 3/4 DRGs 7 weeks after microinjection of AAV5- Rbfox1 shRNA or AAV5-Scramble shRNA. n ​= ​10 mice/group. ∗ P ​< ​0.05 vs. male mice microinjected with AAV5-Scramble shRNA. One-way ANOVA followed by Tukey's post hoc test. (b–h) Effect of microinjection of AAV5- Rbfox1 shRNA or AAV5-Scramble shRNA into the unilateral L 3 / 4 DRGs on paw withdrawal frequencies in response to low (0.07 ​g; b, f) and median (0.4 ​g; c, g) force von Frey filament stimuli and paw withdrawal latencies in response to heat (d, h) and cold stimuli (e) on both the ipsilateral (b–e) and contralateral (f–h) sides at different weeks after AAV5 microinjection. n ​= ​10 mice/group. ∗ P ​< ​0.01, ∗∗ P ​< ​0.01 vs . naive mice microinjected with AAV5-Scramble shRNA at the corresponding time points. Two-way repeated measures ANOVA followed by Tukey's post hoc test. (i, j) Effect of unilateral L 3/4 DRGs microinjected with AAV5- Rbfox1 shRNA or AAV5-Scramble shRNA on spontaneous ongoing pain as evaluated by the CPP paradigm. n ​= ​10 mice/group. ∗∗ P ​< ​0.01, according to two-tailed, independent Student's t -test. (k) Effect of unilateral L 3/4 DRG microinjection with AAV5- Rbfox1 shRNA or AAV5-Scramble shRNA on spinal cord dorsal horn neuronal and astrocyte hyperactivities evidenced by increases in the abundance of p-ERK1/2 and GFAP, respectively, in the ipsilateral L 3/4 spinal cord dorsal horn 7 weeks after viral microinjection. n ​= ​10 mice/group. ∗∗ P ​< ​0.01, one-way ANOVA followed by Tukey's post hoc test.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: Produced, shRNA, Two Tailed Test

Journal: Neurotherapeutics

Article Title: Rbfox1 regulates alternative splicing of Nrcam in primary sensory neurons to mediate peripheral nerve injury-induced neuropathic pain

doi: 10.1016/j.neurot.2023.e00309

Figure Lengend Snippet: SNL led to an increase in l Nrcam mRNA expression in injured DRGs. (a) Exon 10 inclusion was increased by SNL, and the rMATS model revealed exon 10 inclusion reads (X and Y) and exclusion reads (Z) for each sample from the SNL and sham groups. (b) SNL drastically decreased exclusion reads (Z), and enhanced inclusion reads (X and Y) of Nrcam exon 10. n ​= ​12 mice/group. ∗∗ P ＜0.01 vs . sham group according to Student's t -test. (c) The lncLevel value increased significantly in the SNL group compared to sham group. The lncLevel value, which indicates “percentage spliced in” denoting the fraction of mRNAs that represent the inclusion isoform. n ​= ​3 biological replicates (12 mice/group). ∗∗ P ​< ​0.01 vs the sham group according to t -test. (d) Schematic of Nrcam gene splicing variants at exon 10, Nrcam pre-mRNA two splicing variants L- Nrcam (inclusion exon 10) and S- Nrcam (exclusion exon 10). (e) Schematic graph showing the position of primers used in PCR reactions and qRT-PCR to examine total Nrcam mRNA and its variants. (f) Typical images (left panels) demonstrating elevated L- Nrcam and diminished S- Nrcam transcripts in injured DRGs after SNL by summary of qPCR densitometric analysis (right graphs). There were significant increases in the level of L- Nrcam mRNA and corresponding decreases in the amount of S -Nrcam mRNA in injured L 4 DRGs at 3, 7, and 14 days after SNL compared with the sham group. n ​= ​12 mice/group. One-way ANOVA followed by Tukey's post hoc test. ∗ P ​< ​0.05, ∗∗ P ​< ​0.01 vs . the corresponding control group (0 days). (g) Sequence containing Rbfox1 binding motif in Nrcam in human, rat, and mouse. Number represents location from intro 10. (h) Cross-link RNA immunoprecipitation assay of Nrcam pre-mRNA sequence. Cultured DRG neurons were infected with AVV5- Rbfox1 and compared with the AAV5- Gfp infected cultured DRG neurons. Rbfox1 binding to regions of Nrcam pre-mRNA was detected by semiquantitative RT-PCR as indicated. NC: negative control (H 2 O). Input: total purified Nrcam mRNA fragments from the cultured DRG neurons. n ​= ​3 biologic repeats/group. ∗∗P ​< ​0.01, according to 2-tailed, unpaired Student's t -test. (i) Levels of Rbfox1 were assessed in mouse cultured DRG neurons following the indicated transductions. n ​= ​4 biological repeats/treatment. One-way ANOVA followed by Tukey's post hoc test. ∗∗P ​< ​0.01 vs Naive. ## P ​< ​0.01 vs the AAV5 Scramble ​+ ​AAV5- Gfp treatment. (j) mRNA expression S- Nrcam and L- Nrcam were examined in mouse cultured DRG neurons under the specified transductions. n ​= ​4 biological repeats/treatment. One-way ANOVA followed by Tukey's post hoc test.

Article Snippet: The membrane was blocked with 5 ​% nonfat milk in Tris-buffered saline containing 0.1 ​% Tween-20 for 1 ​h at room temperature; the membrane was incubated with the following primary antibodies overnight at 4 ​°C: rabbit anti-Rbfox1 (1:1000; Abcam), rabbit anti-Rbfox2 (1:1000; Abcam), rabbit anti-GAPDH (1:2000; Abcam), rabbit anti-total ERK1/2 (1:1000; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2; 1:1000; Abcam), and mouse anti-GFAP (1:1000; Abcam).

Techniques: Expressing, Quantitative RT-PCR, Sequencing, Binding Assay, Immunoprecipitation, Cell Culture, Infection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Purification

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Gene ontologies of RBFOX1 -dependent differentially spliced genes

Article Snippet: Antibodies Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Gene Expression

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: WGCNA in the RBFOX1 knockdown cell line reflects pathways important to neurodevelopment and to autism. For clarity, only the most highly connected module members are shown. Genes with the highest connectivity (i.e. hubs) are indicated in red. (A) Blue module. (B). Yellow module. Differentially expressed ASD genes are in purple.

Article Snippet: Antibodies Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Knockdown

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: RNA sequencing detects altered alternative splicing patterns in PHNP cells with RBFOX1 knockdown. (A) Heat map showing clustering of gene expression among five biological replicates representing three experimental conditions; wild-type (black), RBFOX1 knockdown (shRBFOX1, red) and non-targeting RNA interference (shGFP, green). Analysis is of the top 250 most significant genes, using the Bayes method with a Spearman correction. (B) Analysis of the intronic regions 400 nucleotides upstream and downstream of the alternative exons whose splicing was most significantly affected by RBFOX1 knockdown for the presence of the binding sites for RBFOX1, NOVA1 or PTBP1. Observed sites are shown as well as the number predicted by iterative analysis of an equivalent number of random introns culled from all human genes. Enrichment of the various sites is indicated by gray boxes and arrows. Significance is based on the normal distribution. ns, not significant. A schematic illustration of the predicted effects on alternative splicing based on the location of the RBFOX1-binding site is shown, with downstream sites enhancing and upstream sites repressing exon inclusion. The correlation between RBFOX1-binding site location and splicing changes identified by RNA sequencing in this study is shown. (C) Validation of splicing changes detected by RNA sequencing. Exons are labeled using a sequential annotation based on location within the gene. Genomic coordinates can be found in Supplementary Material, File S1. qRT-PCR or semi-qRT-PCR was used to calculate the ratio of exon inclusion in the RBFOX1 knockdown cells lines when compared with the shGFP control line. A selection of 25 genes is shown with the differential fold change (log base 2) in exon inclusion detected by RNA sequencing shown in red and the observed fold change by RT-PCR shown in blue. Standard error of the mean is indicated by black bars. (D) Comparison of the RBFOX1 gene set with published gene lists. The number of overlapping genes is indicated along with the percentage they represent from each list. Lists were derived from the references indicated and are also shown in Supplementary Material, File S3. Online sources for gene lists include the Genes to Cognition (G2C) database (http://www.genes2cognition.org/), the Organelle DB (http://organelledb.lsi.umich.edu/), the Online Mendelian Inheritance in Man database (http://www.omim.org/), the GeneTests database (http://www.ncbi.nlm.nih.gov/sites/GeneTests/) and the Simons Foundation Autism Research Initiative database (https://sfari.org/). Lists referenced as supplemental are composites of multiple lists derived from the above sources. P-values were determined based on hypergeometric probability. ER, endoplasmic reticulum.

Article Snippet: Antibodies Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: RNA Sequencing, Alternative Splicing, Knockdown, Gene Expression, Binding Assay, Biomarker Discovery, Labeling, Quantitative RT-PCR, Control, Selection, Reverse Transcription Polymerase Chain Reaction, Comparison, Derivative Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Characterization of differential gene expression in RBFOX1 knockdown cells. (A) A selection of 44 genes is shown with the differential fold change (log base 2) in gene expression detected by RNA sequencing shown in red and the observed fold change by qRT-PCR shown in blue. Standard error of the mean is indicated by black bars. (B) Comparison of the RBFOX1 differentially expressed gene set with published gene lists. The number of overlapping genes is indicated along with the percentage they represent from each list. Lists were derived from the references indicated and are also shown in Supplementary Material, File S6. Online sources for gene lists are as described for Figure 2. Lists referenced as supplemental are composites of multiple lists derived from the above sources. P-values were determined based on hypergeometric probability. ER, endoplasmic reticulum.

Article Snippet: Antibodies Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Gene Expression, Knockdown, Selection, RNA Sequencing, Quantitative RT-PCR, Comparison, Derivative Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Characterization of RBFOX1 expression in human brain and fetal-derived neural progenitor cells. (A) A schematic illustration of the RBFOX1 genomic organization is shown. Figure adapted from Underwood et al. (9). Untranslated exons are shown in light gray, translated exons are in white. The brain-specific exon 16 is shown in dark gray, whereas the muscle-specific exon 17 is shown in black. The location of the RNA-binding domain (RRM) is indicated. (B) The expression level of RBFOX1 was assessed by qRT-PCR, with mRNA from PHNP cells differentiated for the indicated times. Primers were directed against exons 8–9 to detect all RBFOX1 isoforms (light gray). Autoregulatory alternative splicing of exon 11 eliminates RNA binding, so primers against exons 9–11 were utilized to detect isoforms with an active RNA-binding domain (dark gray). (C) In situ hybridization was performed with a human fetal brain, age of 19 weeks, using an S35-labeled antisense riboprobe directed against exons 8–13 of RBFOX1. Two representative coronal and sagittal sections are shown. The sense probe is used as a control (lower panels). (D) To quantitate the pattern of RBFOX1 isoforms expressed in the indicated tissues and cell lines, RT-PCR was performed using primers to amplify exons 15–20, which represent the largest region of alternative splicing diversity in the gene. Amplified products were subcloned and sequenced. Total clones are indicated with the representative counts and percentages of the various alternative spliced isoforms. The most highly expressed patterns are highlighted in gray. c, caudate; cp, cortical plate; gz, germinal zone; p, putamen; t, thalamus.

Article Snippet: Antibodies Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing, Derivative Assay, RNA Binding Assay, Quantitative RT-PCR, Alternative Splicing, In Situ Hybridization, Labeling, Control, Reverse Transcription Polymerase Chain Reaction, Amplification, Clone Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: Gene ontologies of RBFOX1 -related differentially expressed genes

Article Snippet: Antibodies Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing, Migration, Transmission Assay

Journal: Human Molecular Genetics

Article Title: RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

doi: 10.1093/hmg/dds240

Figure Lengend Snippet: A model of RBFOX1 function in PHNPs. During neuronal differentiation, RBFOX1 is induced and directly modulates (solid arrows) an RNA splicing network (black box) which in turns coordinately regulates a transcriptional network of additional genes (black box). Downstream genes present within these networks can further modulate either RNA splicing or transcription to generate additional layers of control (dashed arrows). RBFOX1 can further alter its own splicing and downregulate the activity of these networks as indicated. Additional regulatory factors and/or programs can also contribute to the coordinate regulation of these networks (dotted arrows). The number of factors identified in this study at each of the steps is indicated with selected proteins of interest as mediators noted with their predicted regulatory contribution indicated, see text for complete details. The genes within these networks affect a number of key cellular developmental processes that promote neural development and synaptogenesis, leading to the formation of mature neurons. Disruption of these pathways, particularly genes in the transcriptional network, can lead to neurodevelopmental and/or neuropsychiatric phenotypes in humans. The asterisk indicates factors involved in splicing and/or other aspects of RNA-processing.

Article Snippet: Antibodies Primary antibodies were against HA-tag (1:2000, Covance, Princeton, NJ, USA), RBFOX1 (rabbit polyclonal, 1:500, Aviva Systems Biology, San Diego, CA, USA) or β-actin (mouse monoclonal, 1:250 000, Sigma-Aldrich, St Louis, MO, USA).

Techniques: Control, Activity Assay, Disruption

Journal: Neuron

Article Title: Rbfox splicing factors promote neuronal maturation and axon initial segment assembly

doi: 10.1016/j.neuron.2018.01.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-Rbfox1 (anti-Fox1) D8F8 EMD Millipore MABE159 rabbit anti-Rbfox2 (anti-Rbm9) Bethyl Laboratories A300-864A mouse anti-Rbfox3 (anti-NeuN) A60 EMD Millipore MAB377 mouse anti-Map2 AP-20 EMD Millipore MAB3418 rabbit anti-AnkG H-215 Santa Cruz Biotech Sc-28561 mouse anti-AnkG 463 Santa Cruz Biotech Sc-12719 mouse anti-bIV-spectrin Neuromabs N393/76 guinea pig Hb9 Gift from Thomas M. Jessell (Columbia U.)

Techniques: Produced, Recombinant, Software

Journal: Bioscience Reports

Article Title: Rbfox1 expression in amacrine cells is restricted to GABAergic and VGlut3 glycinergic cells

doi: 10.1042/BSR20220497

Figure Lengend Snippet: Primary and secondary antibodies used for immunohistochemistry

Article Snippet: Rbfox1 , Novus Biologicals , NBP2-13169 , Monoclonal , Mouse , 1:200.

Techniques:

Journal: Bioscience Reports

Article Title: Rbfox1 expression in amacrine cells is restricted to GABAergic and VGlut3 glycinergic cells

doi: 10.1042/BSR20220497

Figure Lengend Snippet: ( A ) In the GCL, all Rbpms-positive cells, RGCs, were also stained for Rbfox1 (yellow arrows point at some Rbpms/Rbfox1-positive cells). Red arrows point at some Rbfox1-positive/Rbpms-negative cells that are most likely displaced ACs. ( B ) In the INL, virtually all clabindin-positive cells were also Rbfox1-positive cells (yellow arrows). Very few Rbfox1-immunostained cells appeared to be calbindin-negative (red arrows). In the GCL, Rbfox1 is expressed in calbindin-positive displaced ACs (yellow arrows), as well as in calbindin-negative cells, many of which are RGCs (red arrows). ( C ) Colocalization of Rbfox1 expression with RGCs in whole mount retinas. All Rbpms-positive cells were immunostained with Rbfof1. Rbfox1-positive/Rbpms-negative cells (presumably displaced ACs) are indicated by white arrows. ( D ) A significant overlap in Rbfox1 and calbindin expression within GCL was observed in whole mount retinas (yellow arrows). Rbfox1-positive/calbindin-negative and calbindin-positive/Rbfox1-negative cells are pointed with red and green arrows, respectively. DAPI; 4',6-diamidino-2-phenylindole; GCL; ganglion cell layer, INL; inner nuclear layer, IPL; inner plexiform layer, ONL; outer nuclear layer, OPL; outer plexiform layer.

Article Snippet: Rbfox1 , Novus Biologicals , NBP2-13169 , Monoclonal , Mouse , 1:200.

Techniques: Staining, Expressing

Journal: Bioscience Reports

Article Title: Rbfox1 expression in amacrine cells is restricted to GABAergic and VGlut3 glycinergic cells

doi: 10.1042/BSR20220497

Figure Lengend Snippet: ( A ) Rbfox1 is expressed in very few GlyT1-positve ACs (yellow arrows). Rbfox1-positive/GlyT1-negative and GlyT1-positive/Rbfox1-negative cells are pointed with red and green arrows, respectively. ( B ) Rbfox1 is expressed in sparse population of VGlut3-positve glycinergic ACs (yellow arrows). Red arrows point at Rbfox1-positive cells that not immunostained for GlyT1 (A) or for VGlut3 (B). The boxed areas of the vertical retinal sections are shown at higher magnification.

Article Snippet: Rbfox1 , Novus Biologicals , NBP2-13169 , Monoclonal , Mouse , 1:200.

Techniques:

Journal: Bioscience Reports

Article Title: Rbfox1 expression in amacrine cells is restricted to GABAergic and VGlut3 glycinergic cells

doi: 10.1042/BSR20220497

Figure Lengend Snippet: ( A ) The vast majority GABAergic ACs in the INL were Rbfox1-positive. Some GABA/Rbfox2-positive ACs are pointed with yellow arrows. Rbfox1-negative GABAergic ACs and Rbfox1-positve/GABA-negative cells are pointed with green and red arrows, respectively. ( B ) Rbfox1 is expressed in all cholinergic (ChAT-positive) starburst ACs both in the INL and GCL. ( C ) Extensive overlap of Rbfox1 with NPY expression was also observed (yellow arrows). Very few NPY-positive cells had very faint staining for Rbfox1 (pointed by green arrows).

Article Snippet: Rbfox1 , Novus Biologicals , NBP2-13169 , Monoclonal , Mouse , 1:200.

Techniques: Expressing, Staining