rabbit anti-munc13 synaptic systems 126103  (Synaptic Systems)

Journal: bioRxiv

Article Title: Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction

doi: 10.1101/2025.08.28.672801

Figure Lengend Snippet: TDP-43 loss of function directly leads to cryptic splicing and reduced gene expression A. The levels of transcripts with cryptic exons in AKT3 , CACNA1E , CEP290 , KALRN , KCNQ2 , MNAT1 , RAP1GAP , SETD5 , STXBP5L , and SYT7 were increased upon TDP-43 knockdown. qPCR experiments were performed in iNeurons treated with TDP-43 shRNAs for seven days. The level of GAPDH was used for normalization. The level in control condition was set to 1. Mean ± s.e.m., n=3, unpaired t-test, ** p<0.01, *** p<0.001, **** p<0.0001. B. The total transcript levels of AKT3 , CACNA1E , KALRN , KCNQ2 , RAP1GAP , SETD5 , and SYT7 were decreased upon TDP-43 knockdown while the total transcript level of STXBP5L was increased. qPCR experiments were performed in iNeurons treated with TDP-43 shRNAs for seven days. The level of GAPDH was used for normalization. The level in control condition was set to 1. Mean ± s.e.m., n=3, unpaired t-test, ** p<0.01, *** p<0.001, **** p<0.0001. C. The total protein levels of UNC13A , KALRN , MNAT1 , RAP1GAP , and SYT7 were decreased upon TDP-43 knockdown. Western blotting experiments were performed in iNeurons treated with TDP-43 shRNAs for twelve days. The level of GAPDH or Tubulin was used for normalization. The level in control condition was set to 1. Mean ± s.e.m., n=3, unpaired t-test, *** p<0.001, **** p<0.0001. D. TDP-43 binding sites or UG-rich motifs were detected in the cryptic splicing region of KALRN , KCNQ2 , MNAT1 , RAP1GAP , SETD5 , and SYT7 . Lane 1, RNA-seq track of TDP-43 positive nuclei from FTD/ALS patient brain tissues; Lane 2, RNA-seq track of TDP-43 negative nuclei from FTD/ALS patient brain tissues; Lane 3, CLIP-seq track of TDP-43 in SH-SY5Y cells; Lane 4, percentage of UG (TG) or GU (GT) dinucleotides in 20bp bins, displayed as IGV tracks with a data range of 0.4–1; red rectangle, cryptic exon.

Article Snippet: Primary antibodies used in this study were: TDP-43 (Proteintech, 10782-2-AP), Tubulin (Cell Signaling Technology, 2144S), GAPDH (Sigma-Aldrich, G8795), UNC13A (Proteintech, 68483-1-Ig), KALRN (Proteintech, 19740-1-AP), RAP1GAP (Abcam, ab32373), SYT7 (Thermo Scientific, PA5-52998), MNAT1 (Proteintech, 11719-1-AP).

Techniques: Gene Expression, Knockdown, Control, Western Blot, Binding Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction

doi: 10.1101/2025.08.28.672801

Figure Lengend Snippet: Cryptic splicing targets of TDP-43 are critical for maintaining neuronal activity A. Schematic diagrams show the experimental design of multielectrode array (MEA) recordings upon knockdown of TDP-43 or its cryptic splicing targets in iNeurons in two batches. Batch 1 examined the effects of a reduction in TDP-43, KALRN, RAP1GAP, SYT7, and UNC13A, and Batch 2 examined the effects of a reduction in TDP-43, KCNQ2, MNAT1, and STMN2. Knockdown or scramble shRNAs were administered on Day 21 in both batches. A first evaluation was conducted on Day 40 in Batch 1 and on Day 30 in Batch 2 (T1). A second evaluation was conducted on Day 48 in Batch 1 and on Day 35 in Batch 2 (T2). B. MEA analysis shows decreases in the weighted mean firing rate (spontaneous firing) and the number of bursts (excitability) upon reductions in TDP-43, RAP1GAP, SYT7, KCNQ2, and MNAT1 at T1, as well as decreases in the synchrony index (connectivity) upon reductions in TDP-43, KALRN, RAP1GAP, SYT7, UNC13A, KCNQ2, and MNAT1 at T2. Mean ± s.e.m., n=8, one-way ANOVA, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Primary antibodies used in this study were: TDP-43 (Proteintech, 10782-2-AP), Tubulin (Cell Signaling Technology, 2144S), GAPDH (Sigma-Aldrich, G8795), UNC13A (Proteintech, 68483-1-Ig), KALRN (Proteintech, 19740-1-AP), RAP1GAP (Abcam, ab32373), SYT7 (Thermo Scientific, PA5-52998), MNAT1 (Proteintech, 11719-1-AP).

Techniques: Activity Assay, Knockdown

Journal: bioRxiv

Article Title: Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction

doi: 10.1101/2025.08.28.672801

Figure Lengend Snippet: Inhibiting cryptic splicing of synaptic genes restores the neuronal dysfunction caused by TDP-43 loss A. Schematic diagram shows the model in which individual splicing-inhibiting ASOs can reduce the cryptic splicing level in an individual target, restore its normal gene expression, and eventually ameliorate neuronal dysfunction caused by TDP-43 loss. B. TDP-43 reduction increases the level of transcripts with cryptic exons in KALRN , RAP1GAP , SYT7 , and UNC13A , which is inhibited by adding ASOs targeting each cryptic splicing event individually. qPCR experiments were performed in iNeurons treated with TDP-43 or scramble (SCR) shRNAs for 7 days and cryptic exon-blocking (CE) or non-targeting (NT) ASOs for 9 days. The level of GAPDH was used for normalization. The level in TDP-43 knockdown condition was set to 1. Mean ± s.e.m., n=2-6, unpaired t-test, ** p<0.01. C. TDP-43 reduction decreases the level of total transcripts in KALRN , RAP1GAP , SYT7 , and UNC13A , which is partially reversed by adding ASOs targeting each cryptic splicing event individually. qPCR experiments were performed in iNeurons treated with TDP-43 or scramble (SCR) shRNAs for 7 days and cryptic exon-blocking (CE) or non-targeting (NT) ASOs for 9 days. The level of GAPDH was used for normalization. The level in TDP-43 knockdown condition was set to 1. Mean ± s.e.m., n=2-7, unpaired t-test, * p<0.05, *** p<0.001, **** p<0.0001. D. TDP-43 reduction decreases the level of total proteins in KALRN, RAP1GAP, SYT7, and UNC13A which is ameliorated by adding ASOs targeting each cryptic splicing event individually. Immunoblotting experiments were performed in iNeurons treated with TDP-43 or scramble (SCR) shRNAs for 10 days and cryptic exon-blocking (CE) or non-targeting (NT) ASOs for 12 days. Representative images are shown on the left and quantifications on the right. The level of GAPDH was used for normalization. The level in TDP-43 knockdown condition was set to 1. Mean ± s.e.m., n=2-4, unpaired t-test, ** p<0.01. E. TDP-43 reduction decreases the number of bursts, which is partially reversed by adding ASOs targeting cryptic splicing in KALRN , RAP1GAP , SYT7 , and UNC13A individually. MEA analysis was conducted as shown by the schematic diagram on the top left: co-culture was treated with TDP-43 or scramble (SCR) shRNAs on Day 21 and cryptic exon-blocking (CE) or non-targeting (NT) ASOs on Days 20 and 27; evaluation was conducted on Day 28. Quantifications are shown on the bottom left, mean ± s.e.m., n=3-8, unpaired t-test, * p<0.05, *** p<0.001. Representative raster plots on the right show neuronal activity in a 30-second time frame for each condition. F. TDP-43 reduction decreases the number of bursts, which is reversed by adding a combinations of ASOs targeting cryptic splicing in KALRN , RAP1GAP , SYT7 , and UNC13A . MEA analysis was conducted as shown by the schematic diagram in E. Quantifications are shown on the left, mean ± s.e.m., n=8, unpaired t-test, * p<0.05, ** p<0.01. Representative raster plots on the right show neuronal activity in a 30-second time frame for each condition.

Article Snippet: Primary antibodies used in this study were: TDP-43 (Proteintech, 10782-2-AP), Tubulin (Cell Signaling Technology, 2144S), GAPDH (Sigma-Aldrich, G8795), UNC13A (Proteintech, 68483-1-Ig), KALRN (Proteintech, 19740-1-AP), RAP1GAP (Abcam, ab32373), SYT7 (Thermo Scientific, PA5-52998), MNAT1 (Proteintech, 11719-1-AP).

Techniques: Gene Expression, Blocking Assay, Knockdown, Western Blot, Co-Culture Assay, Activity Assay

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Relative mRNA expression of UNC13B in multiple myeloma cell lines. ( A ) Quantitative real-time PCR (qPCR) analysis of UNC13B mRNA levels in U266, ARD, and RPMI 8226 cell lines. U266 was used as the reference control for relative quantification, and GAPDH served as the internal reference gene. Expression levels were calculated using the 2 -ΔΔCt method and are presented as fold-change relative to U266. The mean ± standard deviation of UNC13B expression was 1.00 ± 0.26 in U266, 3.07 ± 0.44 in ARD, and 1.41 ± 0.26 in RPMI 8226 cells. Overlaid symbols (●, ■, ▲) denote individual independent experiments ( n = 5 per group); different marker shapes are used only for visual distinction and carry no additional meaning. ( B ) Relative UNC13B mRNA expression in ARD cells following different treatments. The Scramble + 25 μM treatment group was used as the reference for comparison. **** indicate p < 0.0001; ns = not significant ( p ≥ 0.05). Data are presented as the mean ± standard deviation (SD, n = 5 independent experiments).

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Quantitative Proteomics, Gene Expression, Standard Deviation, Marker, Comparison

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on cell proliferation. The proliferation rate of ARD cells in the shUNC13B group was significantly inhibited ( p < 0.0001), suggesting that UNC13B expression correlated with the proliferative capacity of ARD cells. **** indicate p < 0.0001; Data are presented as mean ± SD ( n = 3 independent experiments).

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Expressing

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on the clonogenic ability of cells. ( A ) Representative images of colony formation in soft agar. ARD cells stably transduced with either shUNC13B or scrambled shRNA were seeded into 6-well plates (1000 cells per well) in soft agar consisting of a 0.35% agar upper layer over a 0.6% agar base layer. After 14 days of incubation at 37 °C in 5% CO 2 , visible colonies (>50 µm in diameter) were observed under a microscope. ( B ) Quantification of colony numbers. The total number of colonies per well was recorded.Overlaid symbols (●, ■) denote individual independent experiments; different marker shapes are used only for visual distinction and have no additional meaning. Data are presented as the mean ± SD. Statistical analysis was performed using a two-tailed unpaired t -test. ** indicates p < 0.01. Scale bar: 500 µm.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Stable Transfection, Transduction, shRNA, Incubation, Microscopy, Marker, Two Tailed Test

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on the cell cycle in ARD cells. Five days after transduction with shRNA lentiviral vectors, the shUNC13B group showed a reduced fraction of ARD cells in G1 ( p < 0.0001) and an increased fraction in S phase ( p < 0.0001) compared with the Scramble control, with no significant change in G2/M (ns, not significant). Histograms show DNA-content modeling: red areas denote fitted G1 and G2/M peaks, the blue hatched area denotes S phase, and the black line is the measured distribution; ▲ indicates software-generated peak positions (reference only, not used for quantification). **** indicate p < 0.0001.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Transduction, shRNA, Control, Software, Generated

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on apoptosis in ARD cells. ( A ) Flow cytometric analysis of apoptosis using Annexin V-APC staining. The shUNC13B group exhibited a significantly higher percentage of apoptotic ARD cells compared with the scrambled control group ( p < 0.01); Overlaid symbols (●, ■) denote individual independent experiments; different marker shapes are used only for visual distinction and have no additional meaning. ( B ) Western blot analysis of apoptosis- and signaling-related proteins in ARD cells stably transduced with scrambled shRNA or UNC13B shRNA (shUNC13B) for 72 h. Expression levels of PINK1, CDK2, and AKR7A3 were downregulated, while Bim was upregulated, and PKC levels changed only marginally. In the same samples, UNC13B protein expression was markedly reduced, accompanied by substantial increases in cleaved-PARP (~89 kDa), Bax (~21 kDa), and p21 (CDKN1A) levels. GAPDH served as the loading control. ** indicate p < 0.01.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Staining, Control, Marker, Western Blot, Stable Transfection, Transduction, shRNA, Expressing