syn 1 antibody  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Gpr3 mRNA expressions after NGF-, Fsk-, and KCl-mediated neuronal differentiation in PC12 cells (A and B) PC12 cells were induced to differentiate using serum deprivation (0.5% FBS) and stimulation with NGF (50 ng/mL), Fsk (20 μM), or KCl (50 mM). (A) Representative images from PC12 cells stimulated with NGF, Fsk, or KCl at 8, 12, 24, and 48 h after induced differentiation. Scale bars are 50 μm. (B) To evaluate neuronal differentiation induced by various stimuli in PC12 cells, the expression of βIII-tubulin ( Tub-βIII ) and synapsin I ( Syn1 ) was examined. Representative immunofluorescence images of PC12 cells cultured in normal serum-containing medium (15% FBS) or stimulated with NGF, Fsk, or KCl for 48 h are shown. Cells were double-stained with anti-Tub-βIII (red) and anti-SYN1 (green) antibodies. Scale bars are 50 μm. (C) Representative immunoblots showing Tub-βIII and SYN1 protein expression in PC12 cells at 0, 12, 24, and 48 h after stimulation with NGF, Fsk, or KCl. GAPDH is shown as an endogenous loading control. Right margin indicates molecular mass standards (in kilodaltons, kDa). (D and E) Densitometric quantification of time-dependent changes in Tub-βIII (D) and SYN1 (E) protein expression. Data were normalized to GAPDH and are expressed relative to the 0 h value. Data are presented as the mean ± SEM from five independent cultures (biological replicates). ∗∗∗, p < 0.001; ∗∗, p < 0.01 vs. 0 h for each condition. (F) The expression of Gpr3 mRNA was evaluated at 0, 2, 4, 6, 12, 24, 48, and 96 h after induced differentiation, using RT-qPCR. Data represent relative Gpr3 expression (fold change vs. β-actin ) and are presented as the mean ± SEM from 4–6 independent cultures (biological replicates). ∗∗∗ p < 0.001, ∗∗ p < 0.01 and ∗ p < 0.05 vs. 0 h. NGF, neuronal growth factor; Fsk, forskolin; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Source data for this figure are provided in .

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: Expressing, Immunofluorescence, Cell Culture, Staining, Western Blot, Control, Quantitative RT-PCR

Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Induced Gpr3 expression during neuronal differentiation modulates Syn1–3 expression in PC12 cells (A–C) Effects of the suppression of Gpr3 expression on Syn mRNA expression. PC12 cells were transfected with control or Gpr3 siRNA. Twenty-four hours after transfection, differentiation was induced using serum deprivation (0.5% FBS) and NGF (50 ng/mL). Syn1 (A), Syn2 (B), and Syn3 (C) mRNA expression was evaluated using RT-qPCR at 0, 24, 48, and 96 h after inducing differentiation. Data represent relative mRNA expression normalized to β-actin and are presented as the mean ± SEM for each condition (three independent replicates). ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 vs. control at each time point. (D) Effects of the suppression of Nr4a expression on GPR3-mediated Syn1 mRNA expression. PC12 cells were transfected with Nr4a1 siRNA, Nr4a2 siRNA, or Nr4a3 siRNA together with pc-GPR3mAGFL plasmids. For the control, cells were co-transfected with control siRNA and pc-mAGFL (mock). Syn1 mRNA expression was evaluated using RT-qPCR 24 h after transfection. Data represent relative mRNA expression normalized to β-actin and are presented as the mean ± SEM for each condition (three biological replicates). ∗∗∗ p < 0.001 for each indicated group. (E) Primary cerebellar granule neurons (CGNs) were transfected with pc-GPR3mAGFL together with control siRNA, Nr4a1 siRNA, or Nr4a2/3 siRNAs. Syn1 mRNA expression was evaluated using RT-qPCR 24 h after transfection. Gpr3 upregulation significantly increased Syn1 expression, and this effect was abolished by Nr4a1 siRNA, whereas Nr4a2/3 siRNAs showed no significant suppression. Data represent relative mRNA expression normalized to β-actin and are presented as the mean ± SEM for each condition (three biological replicates). ∗∗ p < 0.01 and ∗ p < 0.05 for each indicated group.

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Developmental changes in Gpr3 , Syn1 , and Nr4a family gene expression in primary cortical neurons from wild-type and Gpr3 −/− mice (A–E) Primary cortical neurons were isolated from postnatal day 0–1 (P0–P1) wild-type (WT), and Gpr3 knockout ( Gpr3 −/− ) mice and cultured in vitro for up to 14 days (DIV0–DIV14). mRNA expression levels of Gpr3 (A), Syn1 (B), Nr4a1 (C), Nr4a2 (D), and Nr4a3 (E) were measured by RT-qPCR at DIV0, DIV0.7 (16 h), DIV1, DIV2, DIV4, DIV7, and DIV14. Data represent relative expression normalized to β-actin and are presented as the mean ± SEM for each condition (three biological replicates). ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 vs. WT neurons at each time point.

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: Gene Expression, Isolation, Knock-Out, Cell Culture, In Vitro, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Reduced SYN1-positive puncta in cortical neurons from Gpr3 −/− mice in vitro (A) Staining for SYN1 and Bassoon in cortical neurons obtained from wild-type and Gpr3 −/− mice. Primary neurons were isolated from postnatal day 0–1 (P0–P1) and cultured for 14 days in vitro (DIV14). Neurons were fixed and immunostained with anti-SYN1 (green) and anti-Bassoon (red) antibodies. Representative images are shown. (B) For quantification, neurons were stained with anti-SYN1 (red) and DAPI (blue) at DIV7 and DIV14; representative images are shown. (C) SYN1-positive puncta were quantified within a region 10–25 μm from the nucleus. Quantification was performed using neurons from wild-type ( n = 6) and Gpr3 −/− ( n = 9) mice at DIV7 (left), and from wild-type ( n = 11) and Gpr3 −/− ( n = 9) mice at DIV14 (right) (C). Data are presented as the mean ± SEM for each condition. ∗ p < 0.05 for each indicated group.

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: In Vitro, Staining, Isolation, Cell Culture

Journal: MedComm

Article Title: Therapeutic Effects of Noninvasive Electrical Stimulation in Combination Transplantation of Human Adipose‐Derived Stem Cells‐Derived Dopaminergic Neuron on the Monkey Model of Parkinson's Disease

doi: 10.1002/mco2.70595

Figure Lengend Snippet: The damage to neurons and synapses is reduced after NES and NES‐DN treatment. (A) Heat maps showing changes in apoptosis and neuron death related genes in WT, MPTP, NES, and NES‐DN. (B) Heat maps showing changes in neural and synaptic related genes in WT, MPTP, NES, and NES‐DN. C, Western blot results of protein levels related to neurons and synapses. (D–G) Quantitative statistical results of changes in neuronal and synaptic related protein levels, NeuN (D), PSD95 (E), synapsin‐1 (F), synaptophysin (G). WT ( n = 3), MPTP ( n = 3), NES ( n = 2), and NES‐DN ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: Primary antibodies were used: TH (Invitrogen; PA5‐85167), Vinculin (Sigma; MAB3574), GAPDH (Proteintech; 60004‐1‐Ig), MNF1 (abcam; ab126575), MNF2 (CST; 9482S), OPA1 (CST; 80471S), DRP1 (abcam; ab56788), Fis1 (CST; 32525S), NeuN (abcam; ab177487), PSD95 (CST; 2507S), synapsin‐1 (CST; 5297S), synaptophysin (CST; 25056S), Iba1 (abcam; ab178846), GFAP (Invitrogen; 13‐0300), SERPINA3 (Proteintech; 12192‐1‐AP), NF‐kB p65 (CST; 8242S), IL6 (abcam; ab233551), and TNF‐a (CST; 3707S).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Synaptic and intrinsic membrane defects disrupt early neural network dynamics in Down syndrome

doi: 10.1038/s41467-025-68048-x

Figure Lengend Snippet: A Pie charts showing proportion of disomic (D21) and trisomic (T21) cells exhibiting excitatory postsynaptic currents (EPSCs). B EPSCs were blocked by CNQX (10 μM) and APV (25 μM) and had fast kinetics with no residual GABAergic currents in the presence of glutamatergic blockers. C–E Reduced frequency ( C ) but not amplitude ( D ) and kinetics ( E ) of EPSCs in T21 cells in normal saline. F Pie charts showing that all D21 cells received EPSCs in Mg 2+ -free saline but 10% of T21 cells did not. G Representative EPSC recordings of D21 and T21 cells outside bursts in 0 Mg 2+ . H–J Reduced frequency ( H ) but not amplitude ( I ) or kinetics ( J ) of EPSCs of T21 cells in 0 Mg 2+ saline. K Confocal images and localisation of presynaptic terminal marker synapsin-1 with pan-axonal neurofilament marker SMI-312 and dendritic marker MAP2 counterstained with nuclei marker DAPI in D21 and T21 cells. Scale bars 10 µm. n = 11–59; number of cells/ 3D image stacks. N numbers of 3D image stacks have been depicted in brackets along with p values on graphs; two-tailed unpaired t-test or Mann-Whitney test. Box plots in C, H show median, 25–75% interquartile range and 2-95% whiskers. Bar charts in D, I and K depict means +/- SEM.

Article Snippet: Immunostaining was performed as previously reported , using primary antibodies against MAP2 (Abcam; ab5392; dilution – 1:1000), SMI-312 pan-axonal neurofilament marker cocktail (Biolegend; 837904; 1:1000), PSD95 (Cell Signaling; 3450; 1:400), Synapsin-1 (Cell Signaling; 5297; 1:400), TUBB3 (Biolegend; 802001; 1:5000), Kv4.3 (Proteintech; 25468-1-AP; 1:200), Kv4.2 (Proteintech; 21298-1-AP; 1:200), VGLUT1 (Synaptic Systems; 135 302; 1:1000), Nestin (Abcam; ab6320; 1:200), PAX6 (Biolegend; 901301; 1:200) and SOX2 (Santa Cruz; sc-17320; 1:200).

Techniques: Saline, Marker, Two Tailed Test, MANN-WHITNEY