Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) Using the neuroblastoma Versteeg (top) and SEQC (bottom) patients data-sets in the R2 Genomics Analysis and Visualization Platform ( http://r2.amc.nl ), patients were divided into high (blue) and low (red) CHL1 gene expression groups by median-centered Log2 ratios, and survival curves were generated. Event-free survival (bottom left) and overall survival (right) curves are shown together with patients numbers in parentheses. ( B ) Relative CHL1 expression levels were plotted in patients with and without relapse from the Versteeg (top) and SEQC (bottom) patients data-sets. n = patients number.

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Expressing, Generated

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) CHL1 mRNA level, quantified by q-RT-PCR, was very low in GI-ME-N, SH-EP-21N_mycn_on, LA-N-1, GI-LI-N, IMR-32, SH-EP-21N_mycn_off, SH-EP-2, SK-N-BE2(C); mean-high in HTLA-230, SK-N-SH and SK-N-F1. ( B ) CHL1 protein levels analyzed by Western Blot in the same NB cell lines. Lower numbers indicate densitometric values.

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) IMR-32 cells transiently transfected with pCEFL-CHL1 and lysed 2, 4, 7, 10 and 15 days after transfection were subjected to Western blot analysis and probed with anti-CHL1 antibody. The empty vector (pCEFL) was used as negative control. ( B ) Morphological characteristics of IMR-32 cells wild type (wt) (1, 2); or transfected with pCEFL (3, 4); or with pCEFL-CHL1 (5, 6). Arrows indicate the neurite-like extensions. (Magnification 20× left panels, 40× right panels). ( C ) Protein lysates from IMR-32 cells wt, or transfected with pCEFL, or with pCEFL-CHL1 were probed with anti-MAP2, anti-Beclin1, or anti-LC3 antibodies. ( D ) Protein lysates from HTLA-230 cells wt, or transfected with sh-ns or with sh-CHL1 were probed with anti-CHL1, anti-MAP2, anti-Beclin 1 and anti-LC3 antibodies. Lower numbers indicate densitometric values. ( E ) Morphological characteristics of HTLA-230 cells wt (1, 2), or stably transfected with non-silencing control shRNA (sh-ns) (3, 4) or with silencing CHL1 shRNA plasmid (sh-CHL1) (5, 6). Cells transfected with sh-CHL1 tend to grow in clusters and may form clumps of rounded cells on top of one another. Arrows indicate the neurite-like extensions detectable in cells wt and in controls (Magnification 20× left panels, 40× right panels).

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Transfection, Western Blot, Plasmid Preparation, Negative Control, Stable Transfection, shRNA

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) Protein lysates from IMR-32 cells wt, or transfected with pCEFL or with pCEFL-CHL1 were collected and subjected to GST-PAK pull-down assay and anti-Rac or anti-Cdc42 Western blot analysis. The same protein lysates were blotted again and probed with anti-phospho-p38, anti-phospho-JNK, and anti-phospho-Akt antibodies. Lower numbers indicate densitometric values normalized to each total protein expression. ( B ) IMR-32 wild type or transfected with pCEFL or with pCEFL-CHL1 were subjected to Western blot and probed with anti-PARP antibody to test apoptosis. The level of apoptosis assessed by the cleaved-PARP was greater in IMR-32 cells transfected with pCEFL-CHL1 than in IMR-32 wt or transfected with pCEFL. ( C ) TUNEL-positive cells were examined by fluorescence microscopy (Magnification 60×). Staining with TUNEL (green) revealed chromatin condensation and the typical morphological changes characteristic of apoptosis (arrow). Quantitative analysis of apoptosis was carried out by counting TUNEL-positive and negative cells. Nuclei were counterstained with DAPI (blue) (Three independent experiments ± SD). ( D ) IMR-32 cells wt, or transfected with pCEFL or with pCEFL-CHL1 were plated in 12-well plastic plates and cultured for 4 days. Every day, cells were trypsinized and counted. Data are representative of three independent experiments ± S.D. ( E ) IMR-32 cells wt or transfected with pCEFL or with pCEFL-CHL1 were subjected to the [ 3 H]thymidine incorporation assay. Cells were analyzed with a β-counter to quantify the amount of radioactivity incorporated after 18 hours of incubation (cpm = counts per minute). (Three independent experiments ± S.D). ( F ) Immunofluorescence analysis of proliferating cells using anti-Ki67 (green). Cells were counterstained with DAPI to visualize nuclei (blue). (Magnification 40×). (Three independent experiments ± SD).

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Transfection, Pull Down Assay, Western Blot, Expressing, TUNEL Assay, Fluorescence, Microscopy, Staining, Cell Culture, Thymidine Incorporation Assay, Radioactivity, Incubation, Immunofluorescence

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) Protein lysates from HTLA-230 cells wt, or transfected with sh-ns or with sh-CHL1, were subjected to GST-PAK pull-down assay and anti-Rac or anti-Cdc42 Western blot analysis. The same lysates were blotted again and probed with anti-phospho-p38, anti-phospho-JNK, and anti-phospho-Akt antibodies. Lower numbers indicate densitometric values normalized to each total protein expression. ( B ) HTLA-230 cells wt, or transfected with sh-ns or with sh-CHL1 were cultured for 4 days. Every day cells were trypsinized and counted (Three independent experiments ± S.D). ( C ) HTLA-230 cells wt, or transfected with sh-CHL1 or with sh-ns were subjected to the [3H]thymidine incorporation assay (cpm = counts per minute) (Three independent experiments ± S.D). ( D ) Immunofluorescence analysis of proliferating cells using anti-Ki67 (green). Cells were counterstained with DAPI to visualize nuclei (blue). (Magnification 40×). (Three independent experiments ± SD).

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Transfection, Pull Down Assay, Western Blot, Expressing, Cell Culture, Thymidine Incorporation Assay, Immunofluorescence

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A – B ) Migration assay for IMR-32 cells wt or transfected with pCEFL or with pCEFL-CHL1 (48 or 72 hours after transfection) (A) and for HTLA-230 cells wt or transfected with sh-ns or with sh-CHL1 (B). Cells were seeded on the upper chamber of a transwell insert and 24 hours later, migrated cells were detached from the lower side of the insert, collected and counted (Three independent experiments ± S.D). ( C – D ) Wound healing assay for IMR-32 cells wt or transfected with pCEFL or with pCEFL-CHL1 (C) and for HTLA-230 cells wt or transfected with sh-ns or with sh-CHL1 (D). Cells monolayers were scratched diagonally through the center of each well with a sterile tip (0 h) and photographed every 24 hours for 3 days under the light microscope (Magnification 4×).

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Migration, Transfection, Wound Healing Assay, Light Microscopy

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) IMR-32 cells wt (1, 2), or transfected with pCEFL (3, 4), or with pCEFL-CHL1 (5, 6) were cultured for 15 days. The morphological characteristics of colonies were visualized by light microscope (1, 3, 5) (Magnification 4×), and the colonies were stained with crystal violet for quantification (2, 4, 6). ( B ) IMR-32 cells wt (1), or transfected with pCEFL (2) or with pCEFL-CHL1 (3) were cultured in soft agar for 20 days (Magnification 4×). ( C ) Colonies in HTLA-230 cells wt (1, 2), or transfected with sh-ns (3, 4), or with sh-CHL1 (5, 6) after 15 days culture. ( D ) Colonies in HTLA-230 cells wt (1) or transfected with sh-ns (2) or with sh-CHL1 (3) cultured in soft agar for 20 days. Histograms show mean colony numbers from three independent experiments ± S.D.

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Transfection, Cell Culture, Light Microscopy, Staining

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) Representative images of orthotopic NB tumors formed by IMR-32 cells wt, or transfected with pCEFL-CHL1, or with empty vector pCEFL, after 28 days from implantation. The average volume of tumors formed by IMR-32 cells transfected with pCEFL-CHL1 cells was smaller than that formed by control cells. ( B ) Representative images of orthotopic NB tumors formed by HTLA-230 cells wt, or transfected with sh-CHL1, or with empty vector sh-ns after 21 days from implantation. The average volume of tumors formed by HTLA-230 cells transfected with sh-CHL1 cells was larger than that formed by control cells. After 3 or 4 weeks, all mice were sacrificed and final tumor tissues were photographed. Tumor volumes were recorded with a caliper and were calculated according to the formula volume = π/6[ω 1 x(ω 2 ) 2 ]. The significance of differences between experimental groups and controls was determined by the unpaired t -test. ( C ) CHL1 protein located on the cell surface (green) as assessed by immunofluorescence with specific mAb in orthotopic tumors formed by IMR-32 cells wt (1) or transfected with pCEFL-CHL1 (2) or by HTLA-230 cells wt (3) or transfected with sh-CHL1 (4) (Magnification 40×). ( D ) Representative images showing the morphological appearance of the orthotopic tumors stained with hematoxylin/eosin. NB tumors developed by implantation of IMR-32 cells wt (1) or transfected with pCEFL-CHL1. The inset shows an enlargement of some differentiating cells (arrow) (2). NB tumors formed by implantation of HTLA-230 cells wt (3) or transfected with sh-CHL1 (4).

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Transfection, Plasmid Preparation, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: ( A ) Immunohistochemistry analysis of MAP2 expression in orthotopic NB obtained by implantation of IMR-32 cells wt or transfected with pCEFL-CHL1. ( B ) Apoptotic cells in NB formed by implantation of IMR-32 cells wt or transfected with pCEFL-CHL1. TUNEL-positive apoptotic cells were detected by localized green fluorescence within cell nuclei counterstained with DAPI (blue) (Magnification 40×). ( C ) MAP2 immunohistochemical staining in NB obtained by implantation of HTLA-230 cells wt or transfected with sh-CHL1. ( D ) Ki67 immunohistochemical staining in NB formed by implantation of HTLA-230 cells wt or transfected with sh-CHL1. The histograms represent the percentage of MAP2 + , Ki67 + and TUNEL + cells.

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Immunohistochemistry, Expressing, Transfection, TUNEL Assay, Fluorescence, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: CHL1 gene acts as a tumor suppressor in human neuroblastoma

doi: 10.18632/oncotarget.25403

Figure Lengend Snippet: In this model, CHL1 binds to yet unknown ligands and inhibits the activation of Rho GTPases, of related p38/JNK MAPK pathways, and of p-Akt. These events, in turn, result into enhanced tumor cell apoptosis and autophagy, differentiation, adhesion and neurite outgrowth, as well as into dampened cell proliferation and motility and tumor progression.

Article Snippet: The following antibodies were used: anti-Ki67 (diluted 1:100, Abcam) and anti-CHL1 (diluted 1:50, Abcam).

Techniques: Activation Assay

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: CHL1 expression in lung tumors from EML4‐ALK transgenic mice. A, EML4‐ALK transgenic mouse lung cancers ( Arrows ). Two representative tumor formations in the lung (upper panel) and HE staining of cancer tissue (lower panel; indicated as the dotted area of “T”). Scale bar: 100 μm. B, RT‐PCR analyses of Gjb4 , MMP13 , CHL1 , Claudin2 and EML4‐ALK mRNA show potent expression in lung cancer tissue. Tissues derived from 12 and 24‐wk old male and female mice were used for the analysis, respectively. N, normal tissue; T, tumor tissue. C, Immunohistochemical staining of lung tissues from EML4‐ALK transgenic mouse. CHL1 staining (upper panel) and Claudin2 staining (lower panel) were performed using anti–CHL1 and anti–Claudin2 antibodies with DIC images. The dotted area indicates the tumor tissue ( T ). D, Protein expression of CHL1 in cancer tissue. Tissue lysate from lung cancer tissue and normal tissue were subjected to western blot analysis using mouse CHL1 antibody. N, normal tissue; T, tumor tissue

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Expressing, Transgenic Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Immunohistochemical staining, Western Blot

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: BiCAT analysis for cultured cancer cells. A, Representative image of EML4‐ALK primary cells. B, C, Partner molecules with CHL1 in EML4‐ALK primary cells were labeled with fluorescein‐arylazide (B) and fluorescein‐tyramide (C) reagent. Enzyme‐mediated activation of radical source (EMARS) products were, respectively, subjected to western blot analysis followed by staining using anti–fluorescein antibody. “CTxB” indicates the positive control sample using CTxB probe, “CHL1” the samples using CHL1 probe, and “(−)” the negative control samples (no probe). D, EMARS products labeled with fluorescein‐tyramide in LK2 cells. Protein expression level of CHL1 in LK2 and RERF cells (left column). EMARS products by CTxB and human CHL1 probes (right column). Abbreviations are the same as in (C). E, Human receptor tyrosine kinase (RTK) antibody array analysis of EMARS products from LK2 cells. EMARS samples were applied to Human RTK antibody array according to the manufacturer's instructions. “CHL1 probe (+)” indicates the sample using CHL1 probe, and “CHL1 probe (−)” the negative control samples (no probe). The proteins corresponding to positive RTK were indicated in the array data. F, Interaction between FGFR3 and α2 integrin in HEK293 cells. HEK293 (mock) and CHL1 transfectant (hCHL1) cells were subjected to western blot analysis with anti–CHL1 antibody (left panel). The EMARS products by HRP‐conjugated anti CHL1 antibody from HEK293 and CHL1 transfectant cells were subjected to 10% SDS‐PAGE gel followed by direct fluorescein detection (middle panel). Immunoprecipitation experiment of fluorescein‐labeled α2 integrin using anti–fluorescein‐Sepharose (right panel). The immunoprecipitation samples and input lysate were subjected to 6% SDS‐PAGE gel followed by the western blot analysis with anti–α2 integrin antibody

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Cell Culture, Labeling, Activation Assay, Western Blot, Staining, Positive Control, Negative Control, Expressing, Ab Array, Transfection, SDS Page, Immunoprecipitation

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: BiCAT located in lung cancer cell membranes and cellular vesicles. A‐C, Morphological observation of BiCAT in LK2 cells using electron microscopy. Cultured LK2 cells were fixed and co–stained with CHL1 (indicated as 10 nm particles) and partner molecules identified in cell membrane. α2 integrin (A), FGFR3 (B) and contactin1 (C) were indicated as 5 nm particles. Arrows indicate the locations of gold particles. Scale bar: 100‐500 nm

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Electron Microscopy, Cell Culture, Staining, Membrane

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: BiCAT located in the pathological specimens from lung cancer patients. A, Representative images of CHL1‐α2 integrin BiCAT‐positive specimens from 55 cases of lung cancer patients. The lung cancer specimens were co–stained with anti–CHL1 antibody (red) and anti–α2 integrin antibody (green), respectively. DAPI solution was used for the nuclear DNA staining. Then, the resulting specimens were observed with confocal microscopy (×5 objective). Both tumor tissues (upper panel) and normal tissue (middle panel) were stained under the same conditions. Representative images at high magnification observation (×20 objective; lower panel) in part of the positive region of BiCAT indicated as the merged area (yellow). B, C, Quantitative analysis of co–expression signals of CHL1‐α2 integrin BiCAT molecule. The co–expression area was quantified using Image J software as described in the Supporting Materials and Methods (Appendix ). B, Tumor slices. C, Normal slices. The quantitative values of the co–expression signals are shown in mean gray value. D, Statistical analysis of mean gray value of CHL1‐α2 integrin BiCAT between normal and tumor tissues. The analysis was performed with the Mann–Whitney test using R software and EZR. P < 1 × 10 −7 . The CHL1‐α2 integrin BiCAT had significantly higher expression in tumor tissues

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: Staining, Confocal Microscopy, Expressing, Software, MANN-WHITNEY

Journal: Cancer Science

Article Title: Proximity proteomics identifies cancer cell membrane cis ‐molecular complex as a potential cancer target

doi: 10.1111/cas.14108

Figure Lengend Snippet: In vitro simulation of effective drug combination to inhibit cancer cell proliferation based on BiCAT information. A, The single and double administration under daily treatment protocol (n = 5). The administration timing is indicated by closed triangles. The cell numbers of the treated cells were measured on Day 2 and Day 4. B, The relative ratio (% of non–treated cells as control) of cell proliferation rates in LK2 cells and EML4‐ALK primary cells. The statistical analysis was performed using Tukey's test and Dunnett's multiple test. The results from Dunnett's test are presented in Figure 6; * P < 0.05; ** P < 0.005; ***P < 0.001. C, Double administration of molecular targeted reagents leads to changes in the expression of partner molecules. The samples of single and double administration under daily treatment conditions (3 d) in LK2 cells were subjected to phos‐tag SDS‐PAGE and then western blot analysis using CHL1, α2 integrin and FGFR3 antibodies. The CBB staining image indicates load control. The molecular weight markers were not shown in this figure because phos‐tag SDS‐PAGE cannot show the correct molecular weight of sample proteins. D, Western blot analysis of phosphorylated FGFR3 in single and double administration samples. The samples under daily treatment conditions (3 d) in LK2 cells were subjected to normal SDS‐PAGE gel and then western blot analysis using anti–FGFR3 and anti–phospho‐FGFR3 antibodies. The quantification of the phosphorylated bands detected in FGFR3 blots was performed using Image J software (ver. 1.51). The ratio of phosphorylation among the samples is indicated below the figure. E, Western blot analysis of phospho‐focal adhesion kinase (FAK) in single and double administration samples. The samples under daily treatment conditions (3 d) in LK2 cells were subjected to normal SDS‐PAGE and then western blot analysis using anti–FAK and anti–phospho‐FAK antibodies

Article Snippet: Briefly, EML4‐ALK primary cells, LK2 cells, HEK293 cells and CHL1 transfectant HEK293 cells were washed once with PBS at room temperature and then treated with either 5 μg/mL of HRP‐conjugated anti–mouse CHL1 antibody (AF2147; R&D systems) and anti–human CHL1 antibody (MAB2126; R&D systems) or 4 μg/mL of HRP‐conjugated CTxB (LIST Biological Laboratories) in PBS at room temperature for 20 minutes.

Techniques: In Vitro, Control, Expressing, SDS Page, Western Blot, Staining, Molecular Weight, Software, Phospho-proteomics

Journal: International journal of molecular sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons.

doi: 10.3390/ijms231710027

Figure Lengend Snippet: Figure 2. NCAM140-ICD and NCAM180-ICD directly bind to the N-terminal ICDs of TRPC1, −4, and −5. Recombinant N-terminal ICDs of TRPC1 (TRPC1-ICDNT) (a,d), TRPC4 (TRPC4-ICDNT) (b,e), and TRPC5 (TRPC5-ICDNT) (c,f) were substrate-coated and incubated with increasing concentrations of recombinant NCAM140-ICD (a–c), NCAM180-ICD (a–c), CHL1-ICD (a–c), or NCAM140-ICD with a deletion of the N-terminal (NCAM140∆N), middle (NCAM140∆M), or C-terminal (NCAM140∆C) parts (d–f). Binding was determined by ELISA using NCAM antibody P61 (a–c), CHL1 antibody (a–c), and NCAM antibody GTX133217 (d–f). Mean values ± SEM from 3 independent experiments carried out in triplicates are shown.

Article Snippet: Polyclonal rabbit TRPC1 antibody H-105 (sc-20110) against amino acids 689–793 at the cytoplasmic C-terminus of human TRPC1; polyclonal rabbit TRPC4/5 antibody H-80 (sc-28760), which is raised against amino acids 1–80 at the cytoplasmic N-terminal domain of human TRPC5 and recognizes TRPC4 and −5; polyclonal rabbit TRPC3/6/7 antibody H-100 (sc-20111), which is raised against amino acids 1–100 at the cytoplasmic N-terminus of human TRPC3 and recognizes TRPC3, −6, and −7; monoclonal mouse TRPC1 antibody E-6 (sc-133076) against amino acids 689–793 at the cytoplasmic C-terminus of human TRPC1; goat polyclonal antibody C-18 (sc-34986) against the ICD of CHL1; α-tubulin antibody TU-02 (sc-8035); TRPC5 antibody 1C8 (sc-293259); and mouse monoclonal PKC antibody A-3 (sc-17769) were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons.

doi: 10.3390/ijms231710027

Figure Lengend Snippet: Figure 3. Colominic acid/PSA binds to the N-terminal ICDs of TRPC1, −4, and −5, and its binding site overlaps with the binding site of NCAM140-ICD. Recombinant N-terminal ICDs of TRPC1 (TRPC1-ICDNT) (a,d), TRPC4 (TRPC4-ICDNT) (b), TRPC5 (TRPC5-ICDNT) (c), and the TRPC1 peptide (d) were substrate-coated and incubated with increasing concentrations of colominic acid/PSA or chondroitin sulfate. Binding was determined by ELISA with antibodies against PSA or chondroitin sulfate. Mean values ± SEM from 3 independent experiments carried out in triplicates are shown. TRPC1 peptide (e) or N-terminal TRPC1-ICD (TRPC1-ICDNT) (f) were substrate-coated and incubated with increasing concentrations of NCAM140-ICD or CHL1-ICD (e) or with one NCAM140-ICD concentration and different concentrations of TRPC1 peptide (f). Binding was determined by ELISA using NCAM antibody P61 (e,f) and CHL1 antibody C-18 (e). Mean values ± SEM from 3 independent experiments carried out in triplicates are shown.

Article Snippet: Polyclonal rabbit TRPC1 antibody H-105 (sc-20110) against amino acids 689–793 at the cytoplasmic C-terminus of human TRPC1; polyclonal rabbit TRPC4/5 antibody H-80 (sc-28760), which is raised against amino acids 1–80 at the cytoplasmic N-terminal domain of human TRPC5 and recognizes TRPC4 and −5; polyclonal rabbit TRPC3/6/7 antibody H-100 (sc-20111), which is raised against amino acids 1–100 at the cytoplasmic N-terminus of human TRPC3 and recognizes TRPC3, −6, and −7; monoclonal mouse TRPC1 antibody E-6 (sc-133076) against amino acids 689–793 at the cytoplasmic C-terminus of human TRPC1; goat polyclonal antibody C-18 (sc-34986) against the ICD of CHL1; α-tubulin antibody TU-02 (sc-8035); TRPC5 antibody 1C8 (sc-293259); and mouse monoclonal PKC antibody A-3 (sc-17769) were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Binding Assay, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: International journal of molecular sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons.

doi: 10.3390/ijms231710027

Figure Lengend Snippet: Figure 10. NCAM-mediated signal transduction and neurite outgrowth depend on TRPC1, −4, and −5. (a,b) Hippocampal neurons were maintained on PLL, NCAM-Fc (NCAM), L1-Fc (L1), or CHL1- Fc (CHL1) and treated without (−) (a) or with control antibody (Ig) (b), TRPC inhibitor SKF96365 (a), or function-blocking TRPC1 antibody T1E3 (b). Total neurite lengths per neuron were measured to determine neurite outgrowth. Mean values ± SEM from 3 independent experiments with duplicates are shown for neurite outgrowth relative to neurons without additives (* p < 0.01; one-way ANOVA with Dunn’s multiple comparison test) or relative to vehicle control (§ p < 0.01; one-way ANOVA with Dunn´s multiple comparison test). (c) Cortical neurons from wild-type (NCAM+/+) (c) and NCAM-deficient (NCAM−/−) (d) mice were treated with vehicle alone or together with NCAM antibody (NCAM Ab) or colominic acid/PSA (CA) in the absence (c,d) or presence of Pico145 (Pico), HC-070 (HC), or M084 (c). Total neurite lengths per neuron were measured to determine neurite outgrowth. Mean values ± SEM from 3 independent experiments with duplicates are shown for neurite outgrowth relative to neurons incubated without additives (* p < 0.05, **** p < 0.0001; one-way ANOVA with Kruskal–Wallis post hoc test). (e,f) Wild-type cortical neurons were treated with NCAM antibody in the absence or presence of HC-070 (HC) Pico145 (Pico) or colominic acid/PSA (CA) and subjected to Western blot analysis with antibodies against total and phosphorylated Erk1/2 and PKC and with a γ-adaptin antibody for loading control. Levels of total and phosphorylated Erk1/2 and PKC were determined by Western blot analysis. Mean values ± SEM are shown for the level of phosphorylated Erk1/2 (e) and PKC (f) relative to total Erk1/2 and PKC levels and relative to the values obtained for NCAM antibody-stimulated neurons in the absence of inhibitors (set to 100%) (* p < 0.05; one-way ANOVA with Kruskal–Wallis post hoc test).

Article Snippet: Polyclonal rabbit TRPC1 antibody H-105 (sc-20110) against amino acids 689–793 at the cytoplasmic C-terminus of human TRPC1; polyclonal rabbit TRPC4/5 antibody H-80 (sc-28760), which is raised against amino acids 1–80 at the cytoplasmic N-terminal domain of human TRPC5 and recognizes TRPC4 and −5; polyclonal rabbit TRPC3/6/7 antibody H-100 (sc-20111), which is raised against amino acids 1–100 at the cytoplasmic N-terminus of human TRPC3 and recognizes TRPC3, −6, and −7; monoclonal mouse TRPC1 antibody E-6 (sc-133076) against amino acids 689–793 at the cytoplasmic C-terminus of human TRPC1; goat polyclonal antibody C-18 (sc-34986) against the ICD of CHL1; α-tubulin antibody TU-02 (sc-8035); TRPC5 antibody 1C8 (sc-293259); and mouse monoclonal PKC antibody A-3 (sc-17769) were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Transduction, Control, Blocking Assay, Comparison, Incubation, Western Blot

Journal: Science translational medicine

Article Title: Tau and other proteins found in Alzheimer’s disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans

doi: 10.1126/scitranslmed.aba6334

Figure Lengend Snippet: (A) Scatterplots showing the correlations between n-APLP1 and n-CHL1 (left; n = 316; β = 0.72, P = 9.1 × 10−48), n-APLP1 and md-Tau (middle; n = 316; β = 0.6, P = 1.3 × 10−30), and n-CHL1 and md-Tau (right; n = 316; β = 0.53, P = 1.7 × 10−33) in the CSF of patients with mild to moderate AD. (B) Scatterplots showing the relationships in amyloid-negative (blue circles) and amyloid-positive (red circles) individuals.

Article Snippet: The following antibodies were used for immunoblot: VPS35 (1:1000; ab57632, mouse, Abcam), actin (1:10,000; NB600–535, mouse, Novus Biologicals), albumin (1:1000; NB600–41532, goat, Novus Biologicals), albumin (1:10,000; ab19194, goat, Abcam), CHL1 (1:500; AF2147-SP, goat, Novus Biologicals), CHL1 (1:1000; 25250–1-AP, rabbit, ProteinTech), APLP1 (1:500; AF3179-SP, rabbit, R&D Systems), Tau (1:500; ab80579, mouse, Abcam), beta-III tubulin (1:5000; ab107216, chicken, Abcam), PSD95 (1:1000; MAB1596, mouse, Millipore), synaptophysin (1:30,000; MAB5258, mouse, Millipore), anti-mouse horseradish peroxidase (HRP) (170–6516, Bio-Rad), anti-goat HRP (1:3000; 172–1034, rabbit, Bio-Rad), anti-rabbit HRP (1706515, goat, Bio-Rad), anti-chicken 800CW (1:20,000; 925–32218, donkey, LI-COR), anti-mouse 680LT (1:20,000; 925–68022, donkey, LI-COR), anti-rabbit 800CW (1:20,000; 925–32211, goat, LI-COR), protein G-HRP (1:10,000; 18–161, Sigma-Aldrich), and protein A-HRP (1:10,000; 101023, Thermo Fisher).

Techniques:

Journal: Science translational medicine

Article Title: Tau and other proteins found in Alzheimer’s disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans

doi: 10.1126/scitranslmed.aba6334

Figure Lengend Snippet: (A) Scatterplots showing the relationship between n-APLP1 and p-tau217 in the CSF of patients with mild to moderate AD (left; n = 316; β = 0.36, P = 6.5 × 10−11), healthy controls (middle; n = 37; β = 0.72, P = 5.1 × 10−7), and MCIs adjusted for CSF Aβ42 (right; n = 21; β = 0.63, P = 0.002). (B) Scatterplots showing relationship between n-CHL1 and p-tau217 in the CSF of patients with mild to moderate AD (left; n = 316; β = 0.37, P = 6.4 × 10−11), healthy controls (middle; n = 37; β = 0.62, P = 5.0 × 10−6), and MCIs adjusted for CSF Aβ42 (right; n = 21; β = 0.71, P = 0.0003).

Article Snippet: The following antibodies were used for immunoblot: VPS35 (1:1000; ab57632, mouse, Abcam), actin (1:10,000; NB600–535, mouse, Novus Biologicals), albumin (1:1000; NB600–41532, goat, Novus Biologicals), albumin (1:10,000; ab19194, goat, Abcam), CHL1 (1:500; AF2147-SP, goat, Novus Biologicals), CHL1 (1:1000; 25250–1-AP, rabbit, ProteinTech), APLP1 (1:500; AF3179-SP, rabbit, R&D Systems), Tau (1:500; ab80579, mouse, Abcam), beta-III tubulin (1:5000; ab107216, chicken, Abcam), PSD95 (1:1000; MAB1596, mouse, Millipore), synaptophysin (1:30,000; MAB5258, mouse, Millipore), anti-mouse horseradish peroxidase (HRP) (170–6516, Bio-Rad), anti-goat HRP (1:3000; 172–1034, rabbit, Bio-Rad), anti-rabbit HRP (1706515, goat, Bio-Rad), anti-chicken 800CW (1:20,000; 925–32218, donkey, LI-COR), anti-mouse 680LT (1:20,000; 925–68022, donkey, LI-COR), anti-rabbit 800CW (1:20,000; 925–32211, goat, LI-COR), protein G-HRP (1:10,000; 18–161, Sigma-Aldrich), and protein A-HRP (1:10,000; 101023, Thermo Fisher).

Techniques:

Journal: Science translational medicine

Article Title: Tau and other proteins found in Alzheimer’s disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans

doi: 10.1126/scitranslmed.aba6334

Figure Lengend Snippet: A set of 39 healthy controls and 19 prodromal AD classified on the basis of Tau/Aβ42 cutoff for AD (≥0.15) were analyzed using the n-APLP1, n-CHL1, and md-Tau assays. Shown is an in-between group comparison analysis in the corrected concentrations of n-APLP1 (F = 84.2, P = 9.4 × 10−13) and n-CHL1 (F = 78.2, P = 3.2 × 10−12) in patients with prodromal AD versus healthy controls.

Article Snippet: The following antibodies were used for immunoblot: VPS35 (1:1000; ab57632, mouse, Abcam), actin (1:10,000; NB600–535, mouse, Novus Biologicals), albumin (1:1000; NB600–41532, goat, Novus Biologicals), albumin (1:10,000; ab19194, goat, Abcam), CHL1 (1:500; AF2147-SP, goat, Novus Biologicals), CHL1 (1:1000; 25250–1-AP, rabbit, ProteinTech), APLP1 (1:500; AF3179-SP, rabbit, R&D Systems), Tau (1:500; ab80579, mouse, Abcam), beta-III tubulin (1:5000; ab107216, chicken, Abcam), PSD95 (1:1000; MAB1596, mouse, Millipore), synaptophysin (1:30,000; MAB5258, mouse, Millipore), anti-mouse horseradish peroxidase (HRP) (170–6516, Bio-Rad), anti-goat HRP (1:3000; 172–1034, rabbit, Bio-Rad), anti-rabbit HRP (1706515, goat, Bio-Rad), anti-chicken 800CW (1:20,000; 925–32218, donkey, LI-COR), anti-mouse 680LT (1:20,000; 925–68022, donkey, LI-COR), anti-rabbit 800CW (1:20,000; 925–32211, goat, LI-COR), protein G-HRP (1:10,000; 18–161, Sigma-Aldrich), and protein A-HRP (1:10,000; 101023, Thermo Fisher).

Techniques: Comparison

Journal: Science translational medicine

Article Title: Tau and other proteins found in Alzheimer’s disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans

doi: 10.1126/scitranslmed.aba6334

Figure Lengend Snippet: (A) Scatterplots showing the relationship between n-APLP1 and n-CHL1 (top; n = 40; β = 0.97, P = 2.4 × 10−24), n-APLP1 and md-Tau (middle; n = 40; β = 0.86, P = 1.7 × 10−12), and n-CHL1 and md-Tau (bottom; n = 40; β = 0.87, P = 2.1 × 10−17) in the CSF of healthy controls. (B) Scatterplot showing the relationship between n-APLP1 and n-CHL1 (top; n = 21; β = 0.97, P = 6.5 × 10−13) in the CSF of patients with MCI. (C) Scatterplot showing relationships between n-APLP1 and n-CHL1 (top: healthy control, blue circles; MCIs, red circles) after adjustment for CSF Aβ42. Scatterplot showing relationships between n-APLP1 and md-Tau (middle; n = 21; β = 0.67, P = 0.001) and n-CHL1 and md-Tau (bottom; n = 21; β = 0.79, P = 0.00002) after adjustment for CSF Aβ42.

Article Snippet: The following antibodies were used for immunoblot: VPS35 (1:1000; ab57632, mouse, Abcam), actin (1:10,000; NB600–535, mouse, Novus Biologicals), albumin (1:1000; NB600–41532, goat, Novus Biologicals), albumin (1:10,000; ab19194, goat, Abcam), CHL1 (1:500; AF2147-SP, goat, Novus Biologicals), CHL1 (1:1000; 25250–1-AP, rabbit, ProteinTech), APLP1 (1:500; AF3179-SP, rabbit, R&D Systems), Tau (1:500; ab80579, mouse, Abcam), beta-III tubulin (1:5000; ab107216, chicken, Abcam), PSD95 (1:1000; MAB1596, mouse, Millipore), synaptophysin (1:30,000; MAB5258, mouse, Millipore), anti-mouse horseradish peroxidase (HRP) (170–6516, Bio-Rad), anti-goat HRP (1:3000; 172–1034, rabbit, Bio-Rad), anti-rabbit HRP (1706515, goat, Bio-Rad), anti-chicken 800CW (1:20,000; 925–32218, donkey, LI-COR), anti-mouse 680LT (1:20,000; 925–68022, donkey, LI-COR), anti-rabbit 800CW (1:20,000; 925–32211, goat, LI-COR), protein G-HRP (1:10,000; 18–161, Sigma-Aldrich), and protein A-HRP (1:10,000; 101023, Thermo Fisher).

Techniques: Control

Journal: Science translational medicine

Article Title: Tau and other proteins found in Alzheimer’s disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans

doi: 10.1126/scitranslmed.aba6334

Figure Lengend Snippet: (A) Immunoblotting with antibodies directed against the N-terminal fragments of APLP1 (n-APLP1) and CHL1 (n-CHL1) in the CSF of a mixed sex cohort of Vps35 cKO mice (red circles) versus controls (black circles) (n = 6 to 8 per genotype; *P < 0.05). Representative immunoblots are shown on the left. Data are shown as means ± SD (middle). Scatterplots showing the relationship between n-APLP1 and n-CHL1 in the CSF of Vps35 cKO mice (red circles) and controls (black circles) (n = 14, β = 0.89, P < 0.0001; right). (B) Simoa assay was used to measure md-Tau in postmortem CSF in a mixed sex cohort of Vps35 cKO mice (red circles) versus controls (black circles) (n = 5 to 9 per genotype; P < 0.00001). Two-tailed unpaired Student’s t test was used for the statistical analysis (left). Antemortem CSF collected from females and males at 3 months (n = 11 to 13 per genotype, P < 0.0001) and 6 months (n = 8 to 10 per genotype, P < 0.0001) of age was analyzed using an md-Tau Simoa assay. A two-way ANOVA Sidak’s multiple comparisons test was used for the statistical analysis (right). Data are shown as means ± SD. (C) Representative Nissl staining analysis in the hippocampus of 3-month-old Vps35 cKO mice compared with their control littermates. n = 6 animals per genotype (P = 0.8182, in a nonparametric Mann-Whitney test). Hippocampal CA1 region is shown at higher magnification. Scale bars, 200 μm. (D) Western blot analysis of hippocampal tissue from 3-month-old Vps35 cKO and control littermates (control) showing expression of neurotubulin (P = 0.0001), presynaptic (PSD95, P = 0.8319), or postsynaptic (synaptophysin, P = 0.3558) markers. Data are shown as means ± SEM. n = 6 animals per genotype. Two-tailed unpaired Student’s t test was used for the statistical analysis (*P < 0.05). Significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.005.

Article Snippet: The following antibodies were used for immunoblot: VPS35 (1:1000; ab57632, mouse, Abcam), actin (1:10,000; NB600–535, mouse, Novus Biologicals), albumin (1:1000; NB600–41532, goat, Novus Biologicals), albumin (1:10,000; ab19194, goat, Abcam), CHL1 (1:500; AF2147-SP, goat, Novus Biologicals), CHL1 (1:1000; 25250–1-AP, rabbit, ProteinTech), APLP1 (1:500; AF3179-SP, rabbit, R&D Systems), Tau (1:500; ab80579, mouse, Abcam), beta-III tubulin (1:5000; ab107216, chicken, Abcam), PSD95 (1:1000; MAB1596, mouse, Millipore), synaptophysin (1:30,000; MAB5258, mouse, Millipore), anti-mouse horseradish peroxidase (HRP) (170–6516, Bio-Rad), anti-goat HRP (1:3000; 172–1034, rabbit, Bio-Rad), anti-rabbit HRP (1706515, goat, Bio-Rad), anti-chicken 800CW (1:20,000; 925–32218, donkey, LI-COR), anti-mouse 680LT (1:20,000; 925–68022, donkey, LI-COR), anti-rabbit 800CW (1:20,000; 925–32211, goat, LI-COR), protein G-HRP (1:10,000; 18–161, Sigma-Aldrich), and protein A-HRP (1:10,000; 101023, Thermo Fisher).

Techniques: Western Blot, Two Tailed Test, Staining, Control, MANN-WHITNEY, Expressing

Journal: Science translational medicine

Article Title: Tau and other proteins found in Alzheimer’s disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans

doi: 10.1126/scitranslmed.aba6334

Figure Lengend Snippet: (A) Listed proteins are those that are significantly reduced or elevated in the CSF of Vps35 cKO mice compared with controls. The four BACE1 substrates are indicated in red. (B) Scatterplots show the distribution of the four identified BACE1 substrates: APLP2, CHL1, APLP1, and APP. Values are reported as label-free quantification (LFQ) intensity and represent the mean of technical duplicates. Bars indicate the means ± SD; Limma-calculated q values are reported (n = 3 to 4 per genotype; ***q < 0.001 and **q < 0.05). (C) As illustrated, these substrates are cleaved at the endosomal membrane by BACE1, liberating N-terminal fragments into the endosomal lumen.

Article Snippet: The following antibodies were used for immunoblot: VPS35 (1:1000; ab57632, mouse, Abcam), actin (1:10,000; NB600–535, mouse, Novus Biologicals), albumin (1:1000; NB600–41532, goat, Novus Biologicals), albumin (1:10,000; ab19194, goat, Abcam), CHL1 (1:500; AF2147-SP, goat, Novus Biologicals), CHL1 (1:1000; 25250–1-AP, rabbit, ProteinTech), APLP1 (1:500; AF3179-SP, rabbit, R&D Systems), Tau (1:500; ab80579, mouse, Abcam), beta-III tubulin (1:5000; ab107216, chicken, Abcam), PSD95 (1:1000; MAB1596, mouse, Millipore), synaptophysin (1:30,000; MAB5258, mouse, Millipore), anti-mouse horseradish peroxidase (HRP) (170–6516, Bio-Rad), anti-goat HRP (1:3000; 172–1034, rabbit, Bio-Rad), anti-rabbit HRP (1706515, goat, Bio-Rad), anti-chicken 800CW (1:20,000; 925–32218, donkey, LI-COR), anti-mouse 680LT (1:20,000; 925–68022, donkey, LI-COR), anti-rabbit 800CW (1:20,000; 925–32211, goat, LI-COR), protein G-HRP (1:10,000; 18–161, Sigma-Aldrich), and protein A-HRP (1:10,000; 101023, Thermo Fisher).

Techniques: Quantitative Proteomics, Membrane

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Expression of CHL1 and NrCAM in pediatric neuroblastoma. Representative examples of CHL1 positive (A) and CHL-1 negative (B) (magnification x100) as well as NrCAM positive (C) and NrCAM negative (D) immunostaining (magnification x200).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing, Immunostaining

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: Kaplan-Meier survival curves for overall and event-free survival. No association was found for CHL1-expression (A/B). Survival rates were better by trend in children with NrCAM positive tumors (C/D) but without statistical significance (p=0.07 and p=0.06).

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing

Journal: Open Medicine

Article Title: CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma

doi: 10.1515/med-2019-0109

Figure Lengend Snippet: CHL1 expression as well as clinical, pathologic and molecular characteristics of the analysed neuroblastoma tissue samples. Statistical analyses by using cross-tables, two-sided Fisher´s and Chi-squared test.

Article Snippet: Afterwards, the primary antibody either specific for CHL1 (goat, polyclonal antibody: AF2126, R&D Systems, MN, USA) or NrCAM (goat anti-human NrCAM antibody: AF2034, R&D Systems, MN, USA,) was applied at 37°C and pH 9.1 for 60 minutes.

Techniques: Expressing, Amplification

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Western blot analysis of CD63 EV marker in precipitated EV samples. The precipitated EVs derived using ExoQuick Solution were prepared from the serum collected from 2 mice of wild-type (WT) and EML4-ALK transgenic (TG) mouse as described in the “ Materials and methods ”. ( B ) Analysis of EMARS products obtained from EMARS reaction for crude mouse serum EVs. EMARS reaction was carried out directly in the precipitated serum EVs from two of WT and TG mice with or without HRP-conjugated CHL1 probe. The EMARS products were subsequently subjected to SDS-PAGE (10% gel) with fluorescein detection and CBB staining. ( C ) Fractionation using Sephacryl S-500 chromatography. Blue dextran (an indicator of void volume) and mouse serum (an indicator of protein elution) were used for preliminary experiments. The dotted line indicates absorbance of blue dextran. The solid line indicates protein concentration measured using a BCA protein kit. ( D ) Morphological observation of serum EVs (fraction No. 6 and No. 8) using cryo-electron microscopy. Lower panel of fraction No.6 is an enlarged view of a part of the upper panel. Scale bar; 200 nm (upper panel) and 50 nm (lower panel).

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Western Blot, Marker, Derivative Assay, Transgenic Assay, SDS Page, Staining, Fractionation, Chromatography, Protein Concentration, Cryo-Electron Microscopy

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Expressing, Transgenic Assay, Western Blot, Membrane

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) The EMARS products for serum EVs from EML4-ALK transgenic mouse. To average experimental results over each group, an aliquot of the serum (10 μL each) from 10 animals in each group (WT), large lung tumor-bearing (TL), and small lung tumor-bearing mice (TS) was mixed in equal proportions, and then applied to EV purification and EMARS. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. The right panel indicates the same gel as the left panel, but exposed for a longer time. ( B ) Confirmation of candidate partner molecules (CD5L and PZP) with mouse CHL1 in EVs. The EMARS products of WT, TL, and TS were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-CD5L (left panel) antibody. After the stripping as described in the “ Materials and methods ”, the membranes were re-stained with anti-PZP antibody (right panel). Arrows indicate the detected band of CD5L and PZP proteins (including predicted dimer). Asterisk indicates unknown bands (predicted as non-specific or partial fragments). ( C, D ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody ( C ). After stripping, the membranes were re-stained with anti-THBS1 antibody ( D ). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( E ) Expression of SLC4A1 (left column) and CHL1 proteins (right column) in tumor tissues from two male and two female EML4-ALK transgenic mice. The fragments of lung cancer tissues were mashed and washed gently with PBS, and then lysed with SDS-PAGE sample buffer directly. The resulting samples were subjected to Western blot analysis with anti-SLC4A1 antibody and anti-CHL1 antibody. Arrows indicate the detected band of monomer SLC4A1 and CHL1 proteins.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Transgenic Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Stripping Membranes, Staining, Expressing

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Western Blot, Molecular Weight, Ubiquitin Proteomics, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Calibration curve of sandwich ELISA for the detection of both SLC4A1 partial proteins and fluorescein-labeled SLC4A1. The detection of several concentrations of recombinant SLC4A1 partial protein using HRP-labeled anti-SLC4A1 antibody which is prepared using Zenon system is summarized in ( A ). The detection of several concentrations of self-made standard materials containing fluorescein-labeled SLC4A1 using HRP-labeled anti-fluorescein antibody is summarized in ( B ). ( C ) Comparison of serum CHL1 levels between wild-type (WT) and small tumor-bearing EML4-ALK transgenic (TS) mice by using previously established ELISA system for CHL1 measurement. There were no significant differences between them.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Sandwich ELISA, Labeling, Recombinant, Comparison, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Calibration curve of sandwich ELISA for the detection of both human CHL1. The detection of several concentrations of recombinant human CHL1 partial protein using HRP-labeled anti-CHL1 antibody. ( B ) Comparison of serum CHL1 levels between H (open bar) and LC (closed bar) by using ELISA system for human CHL1 measurement. There were no significant differences between them. ( C ) Comparison of CHL1-expressing EVs between H (open bar) and LC (closed bar). The serum EVs were purified by using ExoQuick Solution followed by ELISA measurement of CHL1 levels. There were also no significant differences between them.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Sandwich ELISA, Recombinant, Labeling, Comparison, Enzyme-linked Immunosorbent Assay, Expressing, Purification

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) EMARS products purified from serum EVs of healthy person (H) and lung cancer (LC) patients. Fifty microliters of mouse serums was collected from the H and LC groups, and utilized in EV purification followed by EMARS reactions. To average experimental results over each group, an aliquot of the serum (10 μL each) from 5 H and 5 LC was mixed each in equal proportions. The EMARS products were subjected to SDS-PAGE analysis with fluorescence detection. ( B ) Confirmation of caspase 14 as a partner molecule with CHL1 identified by MS proteomics. The H and LC samples were applied respectively to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-caspase 14 antibodies. Arrows indicate the detected band of caspase 14 proteins (including predicted dimer). ( C ) Measurement of fluorescein-labeled caspase 14 using a sandwich ELISA. Serum EVs from 12 H (open bar) and 12 LC (closed bar) were applied to EMARS reactions followed by ELISA measurements, respectively. The EMARS products containing fluorescein-labeled caspase 14 were added to anti-caspase 14 antibody-coated ELISA plates. “BiEV index (caspase 14)” was calculated based on the value of fluorescein-labeled recombinant caspase 14 made by fluorescein-labeling regent. The values are shown as the average of three independent ELISA experiments using the same samples. The detail data of H and LC persons is provided in Table S3. Asterisks indicate the samples were below detection limit. ( D ) ROC curve for BiEV indexes. The AUC was calculated as 0.811. ( E ) Western blot analysis of caspase 14 in whole-serum EVs from H and LC. An aliquot of the serum (2 μL each) from 12 persons in H and LC was mixed in equal proportions followed by EV purification with precipitation protocol. Arrows indicate the detected band of caspase 14.

Article Snippet: The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan).

Techniques: Purification, SDS Page, Fluorescence, Immunoprecipitation, Western Blot, Labeling, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Experimental and Therapeutic Medicine

Article Title: CHL1 inhibits cell proliferation, migration and invasion by regulating the NF‑κB signaling pathway in colorectal cancer

doi: 10.3892/etm.2024.12454

Figure Lengend Snippet: CHL1 inhibits colorectal cancer cell migration and invasion. (A and B) Transwell assay was performed to measure the (A) migratory and (B) invasive ability of HT29 and SW480 cells that were transfected with pcDNA3.1-CHL1 vector (CHL1) and NC. (C) Immunofluorescence labeling of E-cadherin protein in HT29 and SW480 cells. (D) Western blotting was used to detect E-cadherin and N-cadherin protein levels in HT29 and SW480 cells. ** P<0.01 and *** P<0.001 vs. the NC group. CHL1, cell adhesion molecule close homolog of L1; NC, normal control.

Article Snippet: After blocking the membrane with 5% skimmed milk for 1 h at 25˚C, it was incubated overnight with primary antibody E-cadherin (cat. no. 20874-1-AP, Proteintech Group, Inc., dilution 1:20,000), N-cadherin (cat. no. 22018-1-AP, Proteintech Group, Inc., dilution 1:2,000), CHL1 (cat. no. 25250-1-AP, Proteintech Group, Inc., dilution 1:500), p-p65 (cat. no. AP0475; ABclonal, Inc., dilution 1:500), p65 (cat. no. 10745-1-AP, Proteintech Group, Inc., dilution 1:2,000) and GAPDH (cat. no. 10494-1-AP, Proteintech Group, Inc., dilution 1:6,000) at 4˚C.

Techniques: Migration, Transwell Assay, Transfection, Plasmid Preparation, Immunofluorescence, Labeling, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: CHL1 inhibits cell proliferation, migration and invasion by regulating the NF‑κB signaling pathway in colorectal cancer

doi: 10.3892/etm.2024.12454

Figure Lengend Snippet: CHL1 inhibits colorectal cancer cell migration and invasion by regulating the TNF-α/NF-κB pathway. (A and B) Transwell assay was conducted to detect cell migration and invasion. (C) Immunofluorescence assay for positive protein expression of E-cadherin. CHL1 and RELA (p65) overexpression plasmid or NC plasmid were simultaneously transfected for 2 days and then used for this assay. ** P<0.01, and *** P<0.001 vs. the NC group. # P<0.05, ## P<0.01 and ### P<0.001 vs. the CHL1 group. CHL1, cell adhesion molecule close homolog of L1; NC, normal control.

Article Snippet: After blocking the membrane with 5% skimmed milk for 1 h at 25˚C, it was incubated overnight with primary antibody E-cadherin (cat. no. 20874-1-AP, Proteintech Group, Inc., dilution 1:20,000), N-cadherin (cat. no. 22018-1-AP, Proteintech Group, Inc., dilution 1:2,000), CHL1 (cat. no. 25250-1-AP, Proteintech Group, Inc., dilution 1:500), p-p65 (cat. no. AP0475; ABclonal, Inc., dilution 1:500), p65 (cat. no. 10745-1-AP, Proteintech Group, Inc., dilution 1:2,000) and GAPDH (cat. no. 10494-1-AP, Proteintech Group, Inc., dilution 1:6,000) at 4˚C.

Techniques: Migration, Transwell Assay, Immunofluorescence, Expressing, Over Expression, Plasmid Preparation, Transfection