goat against jamc ecd  (R&D Systems)

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: JAM3 is downregulated in LSCC tissues and samples at both mRNA and protein levels. (A) GSE216664, GSE59102 and GSE51985 were used to analyze the different expression levels of JAM3 between N and LSCC T tissues. For GSE216664 and GSE51985, N and T tissues were from the same LSCC patients; for GSE59102, N tissues were ANM tissues from different patients with LSCC. Data are presented as the mean with 95% CI indicated by error bars. **P<0.01, ***P<0.001, ****P<0.0001. (B) Protein levels of JAM3 in the Clinical Proteomic Tumor Analysis Consortium and the International Cancer Proteogenome Consortium datasets among different grades of HNSCC tissues. Data are presented as the mean and range. *P<0.05, **P<0.01, ****P<0.0001 vs. Grade 3. (C) Representative images of JAM3 IHC staining of LSCC tissues and paired ANM tissues. Red scale bar, 150 µm; Black scale bar, 20 µm. (D) IHC results of clinical tissues were analyzed by paired two-tailed Student's t-test. Data are presented as the mean with 95% CI. ****P<0.0001. (E) mRNA expression levels of JAM3 in HaCaT, AMC-HN-8, FD-LSC-1, FaDu and HN30 cell lines. Data are presented as the mean ± SD of three independent experiments. ****P<0.0001 vs. HaCaT. ANM, adjacent normal mucosa; IHC, immunohistochemistry; JAM3, junctional adhesion molecule 3; LSCC, laryngeal squamous cell carcinoma; N, non-tumor; T, tumor.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Expressing, Immunohistochemistry, Two Tailed Test

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Aberrant hypermethylation in the JAM3 promoter is related to low expression of JAM3 . (A) Analysis of JAM3 expression and methylation levels, as well as their correlation using the Gene Expression Omnibus datasets GSE33202 and GSE33205. β value indicates the methylation level of the CpG site. Data are presented as the mean with 95% CI. ***P<0.001, ****P<0.0001. (B) Analysis of JAM3 expression and methylation levels, as well as their correlation in The Cancer Genome Atlas HNSCC samples. β value indicates the methylation level of the CpG site. Data are presented as the mean and range. *P<0.05, ****P<0.0001. (C) Kaplan-Meier plots, generated using the MethSurv webtool highlighted the relationship between methylation levels at certain JAM3 sites and overall survival rates in patients with HNSCC. (D) CpG island (blue area) predicted by MethPrimer tool were detected by bisulfite sequencing PCR in AMC-HN-8 and FD-LSC-1 cell lines. Each row represents an individual cloned allele. Black circles show methylated CpG sites and white circles show unmethylated CpG sites. The percentage methylation rate of each CpG site is shown by the blue-yellow columns; blue indicates unmethylated and yellow indicates methylated sites. (E) Reverse transcription-quantitative PCR and western blotting showing restored expression of JAM3 in both AMC-HN-8 and FD-LSC-1 cells treated with 5-Aza (5 µM) for 72 h. Data are presented as the mean ± SD of three independent experiments. **P<0.01, ***P<0.001 vs. the 5-Aza-group. 5-Aza, 5-Aza-2′-deoxycytidine; HNSCC, head and neck squamous cell carcinoma; JAM3, junctional adhesion molecule 3; FPKM, fragments per kilobase million; LR test, log-likelihood ratio test; HR, hazard ratio.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Expressing, Methylation, Generated, Methylation Sequencing, Clone Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Overexpression of JAM3 suppresses laryngeal squamous cell carcinoma cell proliferation, migration and invasion. (A) AMC-HN-8 and FD-LSC-1 cells were transfected with JAM3 overexpression plasmid or Vec. Expression of JAM3 was examined by reverse transcription-quantitative PCR. Cell proliferation was detected by (B) Cell Counting Kit 8 and (C) colony formation assays. (D) Migration and (E) invasion abilities of AMC-HN-8 and FD-LSC-1 cells after transfection were determined by Transwell assays. Scale bar, 200 µm. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. Vec. JAM3, junctional adhesion molecule 3; Vec, p3×Flag-CMV-10 empty vector.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Knockdown of JAM3 promotes laryngeal squamous cell carcinoma cell proliferation, migration and invasion. (A) AMC-HN-8 and FD-LSC-1 cells were transfected with siRNAs targeting JAM3 (si-JAM3-1, si-JAM3-2) or si-NC. Expression of JAM3 was examined by reverse transcription-quantitative PCR. Cell proliferation was detected by (B) Cell Counting Kit 8 and (C) colony formation assays. (D) Migration and (E) invasion abilities of AMC-HN-8 and FD-LSC-1 cells after transfection were determined by Transwell assays. Scale bar, 200 µm. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the si-NC group. JAM3, junctional adhesion molecule 3; NC, negative control; si, small interfering.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Counting, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: JAM3 mediates laryngeal squamous cell carcinoma tumorigenesis through the Hippo pathway. (A) Protein levels of Flag, JAM3, p-LATS1 (Thr1079), LATS1, p-YAP1 (Ser127), YAP1 and β-actin in AMC-HN-8 and FD-LSC-1 cells with overexpression or knockdown of JAM3 were detected by western blotting. (B) Expression of YAP1 in AMC-HN-8 and FD-LSC-1 cells after transfection with the JAM3 overexpression plasmid or si-JAM3 were detected by confocal microscopy. Scale bar, 50 µm. Fluorescence ratio of YAP1 in the cytoplasm and nucleus of (C) AMC-HN-8 and (D) FD-LSC-1 cells was calculated by ImageJ and analyzed by two-tailed Student's t-test or one-way ANOVA. Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. Vec; # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. si-NC group. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; NC, negative control; p-, phosphorylated; si, small interfering; Vec, p3×Flag-CMV-10 empty vector; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Over Expression, Knockdown, Western Blot, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Two Tailed Test, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Knockdown of JAM3 promotes tumorigenicity of laryngeal squamous cell carcinoma cells in vivo . (A) Representative images of tumors in nude mice after subcutaneous injection of AMC-HN-8 cells transfected with si-NC and si-JAM3. (B) Tumor growth curve was plotted using xenograft tumor volume data. ***P<0.001 vs. si-NC. (C) Tumor weight was measured after tumor excision. *P<0.05 vs. si-NC group. (D) Relative expression levels of JAM3 in xenograft tumors as determined by reverse transcription-quantitative PCR analysis. Data are presented as the mean ± SD of three independent experiments. ****P<0.0001 vs. si-NC. (E) Hematoxylin and eosin staining of xenograft tumors. Immunohistochemistry staining of (F) JAM3, Ki-67 and YAP1, and (G) E-cadherin, N-cadherin and Vimentin in xenograft tumors. Scale bar, 20 µm. JAM3, junctional adhesion molecule 3; NC, negative control; si, small interfering; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Knockdown, In Vivo, Injection, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Immunohistochemistry, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Mechanism by which epigenetic silencing of JAM3 promotes LSCC development by inhibiting the Hippo pathway. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; YAP1, yes-associated protein 1; p, phosphorylated; EMT, epithelial-mesenchymal transition.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques:

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: A Western blot of HEK293T cell lysates transfected with Dcc-pHluorin (Dcc-pH) and JamC-HALO. Immunoprecipitation was performed using GFP or negative control IgG antibodies. Blots were probed for Dcc and HALO-tag, with the yellow arrowhead indicating the expected JamC-HALO band size. B Schematic depicting interactions between Dcc, JamC, and the polarity protein Pard3. Pard3 recruits JamC to the membrane through its PDZ1 domain, which binds the Class 2 PDZ motif at JamC’s C-terminus. JamC interacts with Dcc’s extracellular domain, while Pard3’s PDZ3 the PDZ 3 domain is predicted to interact with a Class 1 PDZ binding motif (X-S/T-X-ϕ COOH ) , , present on Dcc intracellular domain. C , D Airyscan confocal imaging of CGNs nucleofected with Dcc-pHluorin (cyan), JamC-SNAP (yellow), and Halo-Pard3 (magenta). Phluorin and the SNAP dye used here are both pH sensitive highlighting membrane-bound proteins. C Single focal plane showing overlap of Dcc with JamC and Pard3 at the proximal dilation of a CGN (white arrowheads). D Maximum projection of two CGNs forming an adhesion, showing Dcc clustering at the adhesion site before and after Ntn1 addition (200 ng/L). Dcc co-localized with JamC/Pard3 at the adhesion (white arrowhead) and accumulated at the adhesion periphery (hollow arrowhead). Five minutes after the addition of Ntn1 at 200 ng/L, the number of bright Dcc clusters (blue arrowhead) at the membrane surface increased and some newly formed clusters were recruited to the periphery of the JamC/Pard3/Dcc-positive adhesion (white arrowhead). Proximity Labelling Assay (PLA) using Duolink™ fluorescence protocol on fixed dissociated granule neurons plated on laminin and cultured for 24 h, using 2 pairs of primary antibodies: Rabbit against Dcc extracellular domain (ECD) and a Goat against JamC ECD ( E ), and Mouse against Dcc intracellular domain (ICD) and a Rabbit against Pard3 ( F ). Duolink™ staining with no primary, only one or both primaries were compared. Bar graphs represent the ratio of PLA staining intensity (Gray) against Dapi (Cyan) intensity normalized to the negative control without primary antibody (E: n = 4, F: n = 4), replicated 4 (E) and 3 (F) times with similar results. Scale bars: (C, D) 5 µm, (E-F) 10 µm. Error bars represent SEM. See Source Data File.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Western Blot, Transfection, Immunoprecipitation, Negative Control, Membrane, Binding Assay, Imaging, Fluorescence, Cell Culture, Staining

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: A , B Results of ex vivo slice culture assays under different conditions. In each case, the top curve shows the entire distribution of the radial distances of H2B-positive electroporated nuclei from the edge of the slice in replicates. Below this is a plot of the average radial distance from the edge among replicates, and below this is a micrograph representative of the nuclear distribution after 48 h in culture. All are displayed on the same scale, representing a distance from 0 to 300 µm. In addition to H2B-Cherry, the following constructs were electroporated: in ( A ), LacZ+Mir30 shLuc (control, n = 5); LacZ+Mir30 shPard3 ( n = 4); Atoh1::Ntn1+Mir30 shPard3 ( n = 4); Dcc at 0.25 µg+Mir30 shPard3(4); LacZ+Mir30 shJamC ( n = 5); Atoh1::Ntn1+Mir30 shJamC ( n = 5); and Dcc at 0.25 µg+Mir30 shJamC ( n = 3); in ( B ), LacZ+Mir30 shLuc (control, n = 5); Pard3+Mir30 shLuc ( n = 6); Pard3+Mir30 shDcc ( n = 4); JamC+Mir30 shLuc ( n = 3); and JamC+Mir30 shDcc ( n = 4). Each respective control is represented by a red dashed line in the distribution plot. C Chart representing the average variation across replicates in the endpoint nuclear displacement on the x-axis from an even probability of 50%:50%, with negative values representing an attraction to the source of the Ntn1 gradient (in red when statistically significant) and positive values representing repulsion (in blue when statistically significant). Unsorted dissociated CGNs were nucleofected and plated on laminin-coated channel microslides for 24 h, then Ntn1 was added unilaterally into the channel 10 min before the start of nuclear tracking for 2 h. Cells were nucleofected with H2B-mCherry, GPI-pHluorin, and the following: LacZ (control, n = 6), Pard3 ( n = 5), JamC ( n = 7), Mir30 shLuc (control, n = 6), Mir30 shPard3 ( n = 8), and Mir30 shJamC ( n = 6). Abbreviations: EGL external granule layer, ML molecular layer, IGL internal granule layer. In ( A ) through ( C ), error bars represent the SEM. Statistics: ˖ p ≤ 0.1, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, as assessed by an ANOVA followed by a Dunnett post hoc test against the respective controls in (A) and (B) and by a chi-square test in (C) against an even probability of 50%:50%. See Source Data File.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Ex Vivo, Construct, Control

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: A , E Spinning-disk confocal live-cell imaging of dissociated granule neurons plated on laminin for 24 h after nucleofection with Dcc-pHlurorin (Dcc-pH) (cyan), GPI-TdTomato (magenta), and one of the following: in (A), LacZ (Ctrl, n = 10), Siah2 ( n = 14), Pard3 ( n = 14) or JamC ( n = 7); in ( E ), Mir30 shLuc (Ctrl, n = 9), Mir30 shSiah2 ( n = 10), Mir30 shPard3 ( n = 9), or Mir30 shJamC ( n = 9). Cells were tracked for a total of 1 h at 150 s intervals. Representative pictures for each condition show a maximum projection before (t: −1 m 15 s) and after (t: + 6 m 15 s) the addition of Ntn1 at 200 ng/mL. B Schematic representing the segmentation process for the analysis of the time-lapse images in ( A ) and ( E ). The Dcc-pH and GPI-tdTomato channels were segmented using Pixel classification with Ilastik. The resulting “clustered area fraction” is the ratio of the area of the segmented Dcc-pH regions (bright Dcc clusters) to the area of the membrane in each field of view for each time point. C , F Graphs representing the Dcc-pH area fraction over the membrane area, normalized to their respective controls. The different experimental conditions are the same as those in ( A ) and ( E ). A dashed-line marks the addition of 200 ng/mL of Ntn1 at t0. D , G Bar charts highlighting data presented in (C) and (F) for a time point before the addition of Ntn1 (t = −1 m 15 s) and for another time point shortly thereafter (t = +6 m 15 s). In D , G error bars represent the SEM. Statistics: ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, as assessed by an ANOVA followed by a Games–Howell post hoc test against the respective controls. See also Source Data File. Scale bars in (A) and (E) represent 10 µm.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Live Cell Imaging, Membrane

Journal: Nature Communications

Article Title: Siah2 antagonism of Pard3/JamC modulates Ntn1-Dcc signaling to regulate cerebellar granule neuron germinal zone exit

doi: 10.1038/s41467-024-55400-w

Figure Lengend Snippet: The left panel shows the various stages of CGNs and their relative layer occupancy (gray = GNPs, peach = newly differentiated CGN, blue = maturing CGN). The right panel shows the layer-specific response to Netrin-1 produced in the GZ. Siah2 degrades Dcc and Pard3 in GNPs rendering them unresponsive to Netrin-1. Newly differentiated CGNs are slightly attracted to Netrin-1, likely controlling how long these cells remain in the iEGL. Maturing CGNs express high levels of Pard3 which promotes exocytosis of JamC and Dcc and formation of larger structures at the membrane surface. The need of JamC adhesion for Dcc to exit the germinal zone in response to Netrin-1 is at the heart of the coincidence detection circuit.

Article Snippet: Two pairs of primary antibodies were used: Rabbit against Dcc (ECD) (ab273570, Abcam 1:200 dil) and Goat against JamC (ECD) (AF1213, R&D systems, 1:50 dil), and Mouse against Dcc (ICD) (A-1, Santa Cruz 1:100 dil) and Rabbit against Pard3 (07-330, Sigma-Aldrich 1:200 dil).

Techniques: Produced, Membrane

Journal: Neoplasia (New York, N.Y.)

Article Title: Insights into the metastatic bone marrow niche gained from fibronectin and β1 integrin transgenic mice

doi: 10.1016/j.neo.2024.101058

Figure Lengend Snippet: Antibodies used for flow cytometry

Article Snippet: JAM-C , Biotin , Bs-11086R-Biotin , BIOSS , 1:50.

Techniques: