Journal: iScience

Article Title: The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

doi: 10.1016/j.isci.2024.109638

Figure Lengend Snippet: Sema5A increases enteric neurons synapsin 1 phosphorylation at site Ser-603 Western blot analysis of synapsin 1 phosphorylation at site Ser603, Ser9 and Ser62/67 from enteric neuron cultures exposed for 3 days to control Fc, Sema5A-Fc, or Sema5A-S956G-Fc. ∗ p < 0.05 Kruskal-Wallis test, supplemented by Dunn’s post-hoc test. Data are mean ± SEM, n = 8 cultures from 2 independent experiments.

Article Snippet: Membranes were incubated for 1h in blocking solution composed of Tris-buffered saline (TBS), 0.1% (v/v) Tween-20 and 5% (w/v) non-fat powder milk and were then incubated overnight at 4°C in primary antibodies as follows: rabbit polyclonal anti-Sema5A (respectively, 1:500, cat: NBP2-20291, Novus Biologicals and, 1:200, cat: 4460-MG, R&D Systems), rabbit polyclonal anti-Plexin A1 (1:500, cat: APR-081, Alomone), goat polyclonal anti-Plexin A2 (1:500, cat: AF5486, R&D Systems), rabbit monoclonal anti-synapsin 1 (1:1000, cat: 5297, Cell Signaling), mouse monoclonal anti β-tubulin (1:1000, cat: T8660, Sigma-Aldrich), rabbit polyclonal anti-phospho-synapsin 1 (Ser62/67) (1:1000, cat: 11583501, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser603) (1:1000, cat: PA1-4604, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser9) (1:1000, cat: CST2311, Cell Signaling), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (1:1000, cat: 9102, Cell Signaling), rabbit polyclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000, cat: 9101, Cell Signaling), mouse monoclonal anti-CaMKII alpha (1:1000, cat: MA1-048, Invitrogen), rabbit monoclonal anti-phospho-CaMKII (Thr286) (D21E4) (1:1000, cat: 12716, Cell Signaling) and mouse monoclonal anti-β-Actin (1:5000, cat: A5441, Sigma-Aldrich).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: iScience

Article Title: The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

doi: 10.1016/j.isci.2024.109638

Figure Lengend Snippet:

Article Snippet: Membranes were incubated for 1h in blocking solution composed of Tris-buffered saline (TBS), 0.1% (v/v) Tween-20 and 5% (w/v) non-fat powder milk and were then incubated overnight at 4°C in primary antibodies as follows: rabbit polyclonal anti-Sema5A (respectively, 1:500, cat: NBP2-20291, Novus Biologicals and, 1:200, cat: 4460-MG, R&D Systems), rabbit polyclonal anti-Plexin A1 (1:500, cat: APR-081, Alomone), goat polyclonal anti-Plexin A2 (1:500, cat: AF5486, R&D Systems), rabbit monoclonal anti-synapsin 1 (1:1000, cat: 5297, Cell Signaling), mouse monoclonal anti β-tubulin (1:1000, cat: T8660, Sigma-Aldrich), rabbit polyclonal anti-phospho-synapsin 1 (Ser62/67) (1:1000, cat: 11583501, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser603) (1:1000, cat: PA1-4604, Thermo Fisher Scientific), rabbit polyclonal anti-phospho-synapsin 1 (Ser9) (1:1000, cat: CST2311, Cell Signaling), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (1:1000, cat: 9102, Cell Signaling), rabbit polyclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000, cat: 9101, Cell Signaling), mouse monoclonal anti-CaMKII alpha (1:1000, cat: MA1-048, Invitrogen), rabbit monoclonal anti-phospho-CaMKII (Thr286) (D21E4) (1:1000, cat: 12716, Cell Signaling) and mouse monoclonal anti-β-Actin (1:5000, cat: A5441, Sigma-Aldrich).

Techniques: Recombinant, Western Blot, Mutagenesis, Bicinchoninic Acid Protein Assay, Software, Membrane

Journal: iScience

Article Title: The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

doi: 10.1016/j.isci.2024.109638

Figure Lengend Snippet: Sema5A increases enteric neurons synapsin 1 phosphorylation at site Ser-603 Western blot analysis of synapsin 1 phosphorylation at site Ser603, Ser9 and Ser62/67 from enteric neuron cultures exposed for 3 days to control Fc, Sema5A-Fc, or Sema5A-S956G-Fc. ∗ p < 0.05 Kruskal-Wallis test, supplemented by Dunn’s post-hoc test. Data are mean ± SEM, n = 8 cultures from 2 independent experiments.

Article Snippet: Rabbit polyclonal anti-phospho-synapsin 1 (Ser603) , Thermo Fisher Scientific , RRID: AB_560615 ; Cat: PA1-4604.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: iScience

Article Title: The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

doi: 10.1016/j.isci.2024.109638

Figure Lengend Snippet: Enteric neurons exposed to Sema5A for 5 min show increased CaMKII phosphorylation and synapsin 1 phosphorylation at site Ser603 Enteric neurons were treated for 5 min or 30 min with Fc fragment or Sema5A-Fc (4.5 μM). Western blot for phosphorylated CaMKII at Tyr286 (A), phosphorylated ERK at Thr202/Tyr204 (B), and synapsin 1 at Ser-603 (C). ∗ p < 0.05 Mann-Whitney test for each time group. Data are mean ± SEM, n = 12 to 16 cultures from 3 to 4 independent experiments.

Article Snippet: Rabbit polyclonal anti-phospho-synapsin 1 (Ser603) , Thermo Fisher Scientific , RRID: AB_560615 ; Cat: PA1-4604.

Techniques: Phospho-proteomics, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

doi: 10.1016/j.isci.2024.109638

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho-synapsin 1 (Ser603) , Thermo Fisher Scientific , RRID: AB_560615 ; Cat: PA1-4604.

Techniques: Recombinant, Western Blot, Mutagenesis, Bicinchoninic Acid Protein Assay, Software, Membrane