rabbit polyclonal unc13b  (Novus Biologicals)

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 1. Elevated expression of UNC13B in Wilms’ tumor cell lines. (A) Western blot analysis of UNC13B expression in WT‑CLS1, 17.94, G401, SK‑NEP‑1 and HK‑2 cells. Each lane was loaded with 20 µg protein and GAPDH was used as a reference. (B) Analysis of UNC13B transcription levels in different cell lines; n=5. (C) Changes in UNC13B transcription levels 48 h post‑shRNA‑mediated UNC13B knockdown in 17.94 cells compared with the scramble control; n=5. (D) UNC13B expression changes 48 h post‑shRNA‑mediated knockdown in 17.94 cells and (E) statistical analysis of the expression level changes. Each experiment was repeated 3 times, with GAPDH used as a reference. (F) Assessment of cell proliferation post‑knockdown using a Cell Counting Kit‑8 assay, measuring OD450 values at different time points; n=3. Cells were also transfected with non‑target scrambled shRNA as a control. ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin; OD, optical density.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Expressing, Wilms Tumor Assay, Western Blot, Knockdown, Control, CCK-8 Assay, Transfection, shRNA

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 2. UNC13B influences Wilms’ tumor sensitivity to chemotherapy drugs independent of the cell cycle. Evaluation of cell proliferation post‑shRNA‑medi ated UNC13B knockdown after treatment with varying concentrations of (A) vincristine, (B) actinomycin‑D and (C) doxorubicin for 48 h in 17.94 cells, and (D) vincristine, (E) actinomycin‑D and (F) doxorubicin in the G401 cell line, assessed using Cell Counting Kit‑8 assays. Changes in drug sensitivity were analyzed, with dashed lines representing fitted curves for half‑maximal inhibitory concentration calculated using GraphPad software, and the cell number ratio indicating the relative number of viable cells compared between initial cell number and different time points. (G) Cell cycle analysis of 1 µM doxorubicin treatment on control and shUNC13B knockdown cells, detected after 48 h post‑drug treatment. (H) Quantification of the G1, S and G2 phases of the scramble and shUNC13B groups. (I) Typical pseudocolor scatter plots of the apoptosis analysis of UNC13B‑knockdown 17.94 cells after 48 h treatment with 0.5 and 2 µM doxorubicin, and (J) statistical results. **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Wilms Tumor Assay, Knockdown, CCK-8 Assay, Concentration Assay, Software, Cell Cycle Assay, Control

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 3. UNC13B localizes within vesicles and participates in regulating lysosome formation. (A) Indirect immunofluorescence detecting endogenous UNC13B expression in 17.94 cells and revealing UNC13B localization within cellular vesicles. Green fluorescence represents UNC13B and blue fluorescence represents the cell nucleus. (B) Staining of 17.94 cells with Lyso‑Tracker. Cells were cultured in confocal culture dishes. The Mean Gray Value of red fluores cence was calculated in 7 different random fields using ImageJ software. Objective, 20X. (C) Mean Gray Value, calculated by measuring the grayscale values in indicated fluorescence channel regions of interest. ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Cell Culture, Software

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 4. UNC13B modulates cell drug sensitivity by affecting lysosome formation. (A) 17.94 cell line was transiently transfected with the UNC13B‑pCDNA3.1 overexpression vector using Lipofectamine 3,000. Reverse transcription‑quantitative PCR validation was performed 24 h post‑transfection. NC was the trans fection with an empty pcDNA3.1 vector; n=3. (B) UNC13B and LAMP1 expression levels in 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, using GAPDH as a reference, and (C) the associated semi‑quantitative results. (D) Analysis of Mean Gray Value of Lyso‑Tracker in 6 random fields of 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, and (E) representative images of 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells. Lyso‑Tracker indicated lysosomes, as the dye is highly selective for acidic environments, with an excitation wavelength of 577 nm and an emission wavelength of 590 nm. (F) Assessment of doxorubicin sensitivity changes in 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells; n=3. The cell number ratio indicates the relative number of viable cells compared between initial cell number and different time points. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; NC, negative control; OE, over‑expressed; KO, knock‑out; LAMP1, lysosomal‑associated membrane protein 1.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Transfection, Over Expression, Plasmid Preparation, Biomarker Discovery, Expressing, Negative Control, Membrane

Journal: Protein & Cell

Article Title: Sinoatrial node pacemaker cells share dominant biological properties with glutamatergic neurons

doi: 10.1007/s13238-020-00820-9

Figure Lengend Snippet: The SANPCs expressed molecular basis of glutamatergic neurotransmitter system . (A) Expression of functional genes related to glutamate synthesis, reception and transport in SANPCs and CCs in each cluster. The size of the dots showed the percentage of cells which expressed the genes in clusters. The brightness of colors showed the relative expression level of genes. (B and C) Immunofluorescence staining of the expression of glutamatergic and GABAergic neuron markers at the tissue (B) and single cell (C) level. (D and E) Immunofluorescence staining of the expression of the glutamate synthesis marker GLS ( Gls ) and glutamate receptor mGluR5 ( Grm5 ), as well as synaptic vesicle gene UNC13B ( Unc13b ) in SAN tissue (D) and single SANPC (E). Glu, glutamatergic neuron; GABA, GABAergic neuron; OPCs, oligodendrocyte precursor cells; ECs, endothelial cells

Article Snippet: Antibodies included those against GLS (1:25 for immunohistochemistry, 1:100 for immunocytochemistry, ab93434, Abcam), GRINA (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, ab216953, Abcam), GLUR3 (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, ab232887, Abcam), mGluR1 (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, NB110-39033SS, Novus), mGluR5 (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, ab76316, Abcam), VGLUT1 (1:50 for immunohistochemistry, 48-2400, Invitrogen; 1:100 for immunocytochemistry, 135 303, Synaptic Systems), UNC13B (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, TA308990, Origene), SNAP25 (1:50 for immunohistochemistry, GTX113839, Gene Tex; 1:100 for immunocytochemistry, ab31281, Abcam), GAD1 (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, ab26116, Abcam), SYT11 (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, PA5-96970, Invitrogen), CAST (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, PA5-87352, Invitrogen), and HCN4 (1:50 for immunohistochemistry, 1:100 for immunocytochemistry, SAB5200035, Sigma; SMC-320, StressMarq Biosciences).

Techniques: Expressing, Functional Assay, Immunofluorescence, Staining, Marker