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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Western Blot, Marker, Derivative Assay, Transgenic Assay, SDS Page, Staining, Fractionation, Chromatography, Protein Concentration, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Expressing, Transgenic Assay, Western Blot

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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Western Blot, Molecular Weight, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Sandwich ELISA, Labeling, Recombinant, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Sandwich ELISA, Recombinant, Labeling, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

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

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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Western Blot, Marker, Derivative Assay, Transgenic Assay, SDS Page, Staining, Fractionation, Chromatography, Protein Concentration, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Expressing, Transgenic Assay, Western Blot

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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Western Blot, Molecular Weight, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Sandwich ELISA, Labeling, Recombinant, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

Techniques: Sandwich ELISA, Recombinant, Labeling, 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: In human CHL1 ELISA, the recombinant human CHL1 partial protein (10143-H08H; Sino Biological, Beijing, China) was used as a standard.

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

Journal: Scientific Reports

Article Title: BACE1 partial deletion induces synaptic plasticity deficit in adult mice

doi: 10.1038/s41598-019-56329-7

Figure Lengend Snippet: BACE1-mediated processing of APP and CHL1 is reduced in cortex of young BACE1 cKO mice following tamoxifen treatment. Cortex homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for Western blot analysis of APP and CHL1 processing. Homogenates from aged-matched BACE +/− and BACE1 −/− were also loaded as control samples. Representative blots of ( a ) APP-full length (APP-FL) (C1/6.1), ( b ) APP-Carboxy Terminal Fragments (CTFs) (C1/6.1) and ( c ) CHL1. ( d ) Densitometry analysis of protein expression. Protein amount was normalized to protein levels in control mice (set at 1). APP-FL, pC99 and pC89 were normalized to GAPDH (MAB374) while CHL1-FL and CHL1-NTF were normalized to β-tubulin (JDR.3B8). APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the βCTFs pC99 and pC89. βCTFs were clearly identified because missing in the BACE1 −/− sample. CHL1-FL (AF2147) levels were increased while CHL1-N Terminal Fragment (CHL1-NTF) levels were not affected in cortex of TAM-treated mice. However, the CHL1-NTF/CHL1-FL ratio was significantly decreased in TAM-treated mice demonstrating reduced BACE1 processing (VEH n = 8; TAM n = 8). ( e ) Aβx-40 was quantified from brain homogenates by ELISA (VEH n = 8; TAM n = 8). Levels of Aβx-40 expressed as pMol/g of cortex were significantly reduced in TAM-treated mice (~50% decrease). Results were plotted as Mean ± SEM, ***p < 0.001; ****p < 0.0001; n.s. = not significant, Student’s t test.

Article Snippet: Immunoblot and serial fractionation were performed as previously described with the following antibodies: rabbit monoclonal anti-BACE1 (1:1000; D10E5; Cell signaling technology); mouse monoclonal anti-APP (and APP CTFs) antibody (1:5000; C1/6.1; BioLegend); goat polyclonal anti-N-terminal CHL1 antibody (for CHL1-FL and CHL1-NTF) (1:1000; AF2147; R&D Systems); mouse monoclonal anti-GAPDH (1:10,000; MAP374; Millipore); mouse monoclonal anti-β-tubulin (1:10,000; JDR.3BR; Sigma); mouse monoclonal anti-calnexin (1:2000; 610523; BD biosciences); rabbit polyclonal anti-ADAM10 (1:1000;AB19026; Millipore); rabbit polyclonal anti-PS1 AB14 (1:1000) and rat monoclonal anti-SEZ6 (1:250) , rat monoclonal anti-APPsβ (1:40) and HRP-conjugated secondary antibodies visualized by ECL (GE Healthcare).

Techniques: SDS Page, Western Blot, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: BACE1 partial deletion induces synaptic plasticity deficit in adult mice

doi: 10.1038/s41598-019-56329-7

Figure Lengend Snippet: BACE1-mediated processing of APP and CHL1 is reduced in cortex of aged BACE1 cKO mice following tamoxifen treatment. Cortex homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for Western blot analysis of APP and CHL1 processing. Homogenates from aged-matched BACE +/− and BACE1 −/− were also loaded as control samples. APP-FL, pC99 and pC89 were normalized to GAPDH (MAB374) while CHL1-FL and CHL1-NTF were normalized to β-tubulin (JDR.3B8). Protein amount was normalized to protein levels in control mice injected with vehicle (set at 1). Representative blots of ( a ) APP-FL (C1/6.1), ( b ) APP-CTFs (C1/6.1) and (c ) CHL1. ( d ) Densitometry analysis of protein expression. APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the βCTFs pC99 and pC89. βCTFs were clearly identified because missing in the BACE1 −/− sample. CHL1-FL (AF2147) levels were increased and CHL1-NTF levels were significantly reduced. Furthermore, the CHL1-NTF/CHL1-FL ratio was significantly decreased in TAM-treated mice demonstrating reduced BACE1 processing (VEH n = 7; TAM n = 7). ( e ) Quantification of Aβx-40 was performed by MSD immunoassay on cortex homogenates and expressed as pMol/g of cortex. The decrease of levels of Aβx-40 in TAM-treated mice was comparable to the one observed in samples collected from young TAM-treated mice (~50% decrease) (VEH n = 7; TAM n = 7). Results were plotted as Mean ± SEM, *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001; n.s. = not significant, Student’s t test.

Article Snippet: Immunoblot and serial fractionation were performed as previously described with the following antibodies: rabbit monoclonal anti-BACE1 (1:1000; D10E5; Cell signaling technology); mouse monoclonal anti-APP (and APP CTFs) antibody (1:5000; C1/6.1; BioLegend); goat polyclonal anti-N-terminal CHL1 antibody (for CHL1-FL and CHL1-NTF) (1:1000; AF2147; R&D Systems); mouse monoclonal anti-GAPDH (1:10,000; MAP374; Millipore); mouse monoclonal anti-β-tubulin (1:10,000; JDR.3BR; Sigma); mouse monoclonal anti-calnexin (1:2000; 610523; BD biosciences); rabbit polyclonal anti-ADAM10 (1:1000;AB19026; Millipore); rabbit polyclonal anti-PS1 AB14 (1:1000) and rat monoclonal anti-SEZ6 (1:250) , rat monoclonal anti-APPsβ (1:40) and HRP-conjugated secondary antibodies visualized by ECL (GE Healthcare).

Techniques: SDS Page, Western Blot, Control, Injection, Expressing

Journal: Scientific Reports

Article Title: BACE1 partial deletion induces synaptic plasticity deficit in adult mice

doi: 10.1038/s41598-019-56329-7

Figure Lengend Snippet: Axon guidance defects were absent in hippocampus mossy fibers of aged BACE1 cKO mice following partial BACE1 deletion. ( a ) Coronal sections collected from aged mice were stained with anti-synaptoporin (SPO) antibody (green) and DAPI (blue). Scale bar 50 μm. ( b) Quantification of IPB length showed no alteration in TAM-treated mice compared to controls. IPB length was normalized on the length of the CA3 stratum lucidum (VEH n = 8; TAM n = 7, 3 to 4 sections per mouse). ( c ) Representative microscopy images showing reduced BACE1 (D10E5) expression in the hippocampus of TAM-treated mice. BACE1 signal was totally absent in BACE −/− mice, used as control to evaluate the amount of background in the staining. Scale bar 200 μm. Hippocampus full homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for analysis of APP processing and fractionated (soluble and membrane fractions) for the analysis of SEZ6 and CHL1 processing. Homogenates from aged-matched BACE +/− and BACE1 −/− were loaded as control samples. Representative blots of ( d ) APP-FL (C1/6.1) and APP- CTFs (C1/6.1), ( e ) fractionation blots of sAPPβ (BAWT), SEZ6 (14E5) and CHL1 (AF2147). ( f ) Densitometry analysis of protein expression. APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the βCTFs pC99 and pC89, and sAPPβ. βCTFs and sAPPβ were identified because missing in the BACE1 −/− sample. SEZ6 processing was decreased in TAM-treated mice with accumulation of the full length and decreased levels of the ectodomain (SEZ6-NTF) as well as decreased SEZ6-NTF/SEZ6FL ratio. Processing of CHL1 was also impaired as showed by increased of CHL1-FL levels, while CHL1-NTF was not altered. CHL1-NTF/CHL1-FL ratio was significantly decreased. APP-FL, CTFs, SEZ6-NTF and CHL1-NTF were normalized to GAPDH (MAB374), SEZ6-FL and CHL1-FL were normalized to Calnexin (610523) (VEH n = 5; TAM n = 5). ( g ) Aβx-40 was quantified from hippocampus homogenates by MSD immunoassay. TAM-treated group displayed a significant reduction of Aβx-40 levels (~50% decrease) compared to control (VEH n = 7; TAM n = 7). Results were plotted as Mean ± SEM, **p < 0.005; ***p < 0.001; n.s. = not significant, Student’s t test. DG: dentate gyrus, IPB: infrapyramidal bundle, slu: stratum lucidum, MB: main bundle.

Article Snippet: Immunoblot and serial fractionation were performed as previously described with the following antibodies: rabbit monoclonal anti-BACE1 (1:1000; D10E5; Cell signaling technology); mouse monoclonal anti-APP (and APP CTFs) antibody (1:5000; C1/6.1; BioLegend); goat polyclonal anti-N-terminal CHL1 antibody (for CHL1-FL and CHL1-NTF) (1:1000; AF2147; R&D Systems); mouse monoclonal anti-GAPDH (1:10,000; MAP374; Millipore); mouse monoclonal anti-β-tubulin (1:10,000; JDR.3BR; Sigma); mouse monoclonal anti-calnexin (1:2000; 610523; BD biosciences); rabbit polyclonal anti-ADAM10 (1:1000;AB19026; Millipore); rabbit polyclonal anti-PS1 AB14 (1:1000) and rat monoclonal anti-SEZ6 (1:250) , rat monoclonal anti-APPsβ (1:40) and HRP-conjugated secondary antibodies visualized by ECL (GE Healthcare).

Techniques: Staining, Microscopy, Expressing, Control, SDS Page, Membrane, Fractionation

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Sequences for random control siRNA and siRNAs against CHL1.

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Control, Sequencing

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Western blot analysis of the protein levels of CHL1 detected in normal human glial HEB cells and 3 glioma/glioblastoma cell lines. CHL1 was weakly expressed in normal human HEB glial cells. Its levels in all the 3 glioma/glioblastoma cells were higher than that in normal human HEB glial cells, with the statistical significance detected in SHG44 cells (* p < 0.05 vs. HEB cells) and U-87 MG cells (** p < 0.01 vs. HEB cells). n = 3 for each group. Student’s t -test for independent samples was used.

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Treatment of siRNA targeting CHL1 in three human glioma cell lines. Total RNA was isolated from U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). RT-PCR and Western blot analysis were then used to measure both relative mRNA and protein levels of CHL1. (A) RT-PCR analysis of the mRNA levels of CHL1 in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1, and (B) Western blot analysis of the protein levels of CHL1 detected in U251, SHG44 and U-87 MG cells treated with vehicle control (vc), control siRNA and siRNA targeting CHL1. Data are presented as means ± standard error of the mean (SEM) ( n = 3, * p < 0.05; ** p < 0.01, independent Student’s t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 affects the proliferation and survival of U251, SHG44 and U-87 MG glioma cells. Cells were seeded on 96-well plates in triplicate, and proliferation rates were measured by MTT assay to evaluate the effect of CHL1 on the proliferation of U251, SHG44 and U-87 MG glioma/glioblastoma cells. (A–C) Changes of the proliferation rate in U251 (A) , SHG44 (B) , and U-87 MG (C) cells treated with vehicle control (vc), control siRNA (control siRNA) or siRNA targeting CHL1 (CHL1 siRNA). The data were expressed as the means ± SEM of three independent experiments (* p < 0.05 and ** p < 0.01 vs. either vehicle control or control siRNA; independent Student’s t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Knockdown, MTT Assay, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 affects the senescence of glioma/glioblastoma cells in vitro . (A–C) U251 (A) , SHG44 (B) and U-87 MG (C) cells were seeded onto 24-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, and senescent cells were then detected by senescence-associated β-galactosidase staining (200×). The data were expressed as the means ± SEM from four independent experiments (** p < 0.01; *** p < 0.001 vs. either vehicle control or control siRNA; Independent Student’s t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Knockdown, In Vitro, Control, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 reduced colony formation capacity of glioma/glioblastoma cells in vitro . Cell colony was stained by crystal violet, which was then dissolved in 1% SDS and the optical density was measured at 546 nm under a microplate reader. (A) The colony formation assay revealed that knockdown of CHL1 reduced the colony formation of U251 cells, as was revealed by the optical density detected at 546 nm from three independent experiments (* p < 0.05; ** p < 0.01 vs. either vehicle control or control siRNA). (B,C) Similar results were found in SHG44 (B) and U-87 MG (C) cells for the colony formation experiment. The data were expressed as the means ± SEM from 4 independent experiments (* p < 0.05 and ** p < 0.01 vs. both vehicle control and control siRNA; independent Student’s t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Knockdown, In Vitro, Staining, Colony Assay, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 suppressed the migration of glioma/glioblastoma cells in vitro . Transwell migration assays were carried out using U251 (A) , SHG44 (B) and U-87 MG (C) cells transfected with CHL siRNA. Representative fields containing migrated cells attached to the underside of the membrane were presented. The migration ability was indexed by the relative number of migrated cells from three independent experiments. The data were expressed as the means ± SEM from 3 independent experiments (* p < 0.05; ** p < 0.01 vs. both vehicle control and control siRNA (independent Student’s t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Knockdown, Migration, In Vitro, Transfection, Membrane, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 affects apoptosis signaling molecules in glioma/glioblastoma cells. U251, SHG44 and U-87 MG cells were seeded onto 48-well plates and treated with vehicle control, control siRNA and siRNA targeting CHL1, respectively. Western blot analysis was performed to determine the levels the apoptosis-related proteins, including changes of the ratio of Bax to Bcl-2 (Bax/Bcl-2) (A) , active caspase-3 (B) and PCNA (C) in glioma/glioblastoma cells. GAPDH was used as the loading control. The data were expressed as the means ± SEM from three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. either vehicle control or control siRNA; independent Student’s t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Knockdown, Control, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: Knockdown of CHL1 reduced the phosphorylation levels of ERK and AKT. Western blot was used to analyze the levels of pAkt and pErk in three cell lines after treatment with vehicle control, negative control and CHL1 siRNA for 48 h. pAkt and pErk protein levels in U251 (A) , SHG44 (B) and U-87 MG cells (C) were presented. GAPDH was used as a loading control. The data were expressed as the means ± SEM from 3 independent experiments (* p < 0.05 and ** p < 0.01 vs. both vehicle control and control siRNA; independent Students t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Knockdown, Phospho-proteomics, Western Blot, Control, Negative Control

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: CHL1 regulates growth of U-87 MG glioma cells in vivo . (A,B) Two weeks after the 1st intratumoral injection, all mice were killed by cervical dislocation. The in situ tumors and the dissected tumor tissues were photographed. (C) The fold increase of volume at each day points post the 1st intratumoral injection of either control siRNA or CHL1 siRNA complexed with the Entranster™- in vivo . (D) Column diagram showing the final average tumor volumes from both control siRNA and CHL1 siRNA-treated group ( n = 5, p = 0.2768 vs. the control siRNA group) (* p < 0.05; ** p < 0.01 vs. control siRNA; Independent Student’s t -test).

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: In Vivo, Injection, In Situ, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: CHL1 Is Expressed and Functions as a Malignancy Promoter in Glioma Cells

doi: 10.3389/fnmol.2017.00324

Figure Lengend Snippet: H&E staining and immunohistochemical staining analyses for the CHL1, caspase-3, PCNA and GFAP molecules in glioblastoma xenograft tissues from both control siRNA and CHL1 siRNA-treated groups. Scale bars represent 25 μm.

Article Snippet: Then, sections were blocked with 10% normal goat serum in PBS at room temperature for 30 min, and samples were subjected to incubation with the following primary antibodies: rat anti-human CHL1 antibody (1:100, cat. no. MAB2126, R&D Systems), rabbit polyclonal anti-PCNA antibody (1:200, cat. no. sc-7907, Santa Cruz), rabbit polyclonal anti-caspase-3 antibody (1:200, cat. no. sc-7148, Santa Cruz), rabbit polyclonal anti-GFAP antibody (1:500, cat. no. BA0056, Boster Biological Technology) at 4°C overnight.

Techniques: Staining, Immunohistochemical staining, Control

Journal: Development (Cambridge, England)

Article Title: Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development

doi: 10.1242/dev.201811

Figure Lengend Snippet: Endothelial Smad6 maintains embryonic liver vessels via Alk1 regulation. (A-D) Representative images of E16.5 liver sections of indicated genotypes. (A,B) Representative light-sheet images of cleared whole livers stained for Lyve1 and αSMA. (A) Top, overview with boxed areas magnified below. Arrows indicate large veins; arrowhead shows dilated peripheral vessel. (B) Arrowheads indicate ectopic αSMA stain. (C) H&E stain. Far left, whole liver sections. Yellow boxed areas are magnified to the right. Middle, areas of normal liver parenchyma in livers of indicated genotypes. Yellow boxed areas are magnified to right. Far right, areas of abnormal parenchyma in Smad6 mutant liver sections. Yellow boxed areas are magnified to right. Arrows indicate hemorrhage; arrowheads show tissue disorganization. (D) Representative immunofluorescence images stained for PECAM1 (endothelial) and Ter119 (red blood cells) at the periphery of E16.5 livers of indicated genotypes. Scale bars: 500 µm (A, top); 300 µm (A, bottom); 150 µm (B); 20 µm (C); 50 µm (D).

Article Snippet: Livers were rehydrated in a methanol/H 2 O series, rinsed in PBS for 45 min at RT, washed in PTx.2 [0.2% Triton X-100 (Sigma-Aldrich, X100) in PBS] 2× for 30 min at RT, permeabilized in permeabilization solution [0.3 M glycine (Sigma-Aldrich, G7126)+0% DMSO (Fisher D128) in PTx.2] for 1.5 day at 37°C, incubated in blocking solution [6% donkey serum (Sigma-Aldrich, D9663)+10% DMSO in PTx.2] for 1.5 day at 37°C, followed by primary antibody incubation for 3 days at 37°C (Lyve1; R&D Biosystems, AF2126, goat, 0.2 mg/ml stock) diluted 1:100 in PTwH solution [0.2% Tween-20 (Sigma-Aldrich, P9416)+0.01 mg/ml Heparin (Sigma-Aldrich, H3393) in PBS supplemented with 3% donkey serum+5% DMSO].

Techniques: Staining, Mutagenesis, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development

doi: 10.1242/dev.201811

Figure Lengend Snippet: Endothelial Smad6 maintains embryonic liver vascular patterning via Alk1 regulation. (A,B) Representative images of E16.5 liver sections of indicated genotypes. (A) Immunofluorescence for indicated endothelial markers on adjacent sections. Yellow dashed line indicates liver outline; blue dashed line indicates avascular areas. (B) Immunofluorescence for DAPI (nucleus) and Lyve1 (hepatic endothelial cell). Yellow dashed line indicates liver outline; white dashed square shows areas magnified to right. Insets show Lyve1 staining of vascular areas (′) and second areas that are avascular in mutants (″). (C) Quantification of vascularized area (Lyve1 + )/whole liver area. WT, n =11; Smad6 iΔ/iΔ , n =4; Smad6 iΔEC/iΔEC , n =13; Alk1 +/iΔEC , n =7; Smad6 iΔEC/iΔEC ; Alk1 +/iΔEC , n =9 livers. (D) Representative images of immunofluorescence for Lyve1 and cleaved caspase 3. (E) Quantification of whole liver scans of cleaved caspase 3 channel/total region of interest (ROI) area. WT, n =7; Smad6 iΔ/iΔ , n =3; Smad6 iΔEC/iΔEC , n =6; Alk1 +/iΔEC , n =5; Smad6 iΔEC/iΔEC ; Alk1 +/iΔEC , n =6 livers. *** P <0.001; **** P <0.0001; ns, not significant. Data are individual points and mean±s.d. One-way ANOVA with Tukey's multiple comparisons test. Scale bars: 500 µm (A,B); 50 µm (B, insets, D).

Article Snippet: Livers were rehydrated in a methanol/H 2 O series, rinsed in PBS for 45 min at RT, washed in PTx.2 [0.2% Triton X-100 (Sigma-Aldrich, X100) in PBS] 2× for 30 min at RT, permeabilized in permeabilization solution [0.3 M glycine (Sigma-Aldrich, G7126)+0% DMSO (Fisher D128) in PTx.2] for 1.5 day at 37°C, incubated in blocking solution [6% donkey serum (Sigma-Aldrich, D9663)+10% DMSO in PTx.2] for 1.5 day at 37°C, followed by primary antibody incubation for 3 days at 37°C (Lyve1; R&D Biosystems, AF2126, goat, 0.2 mg/ml stock) diluted 1:100 in PTwH solution [0.2% Tween-20 (Sigma-Aldrich, P9416)+0.01 mg/ml Heparin (Sigma-Aldrich, H3393) in PBS supplemented with 3% donkey serum+5% DMSO].

Techniques: Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development

doi: 10.1242/dev.201811

Figure Lengend Snippet: Hepatic endothelial cell Smad6 loss augments LSEC capillarization. (A) Representative images of immunofluorescence staining of E16.5 liver sections of indicated genotypes for Lyve1, collagen IV and αSMA. (B) Collagen IV area/region of interest (ROI) quantification. WT, n =6; Smad6 iΔ/iΔ , n =3; Smad6 iΔEC/iΔEC , n =4; Alk1 +/iΔEC , n =5; Smad6 iΔEC/iΔEC ; Alk1 +/iΔEC , n =6 livers. Data are individual points for each embryo and mean±s.d. One-way ANOVA with Tukey's multiple comparisons test. (C) Quantification of capillary diameter, ≥42 capillaries/embryo measured. WT, n =3; Smad6 iΔ/iΔ , n =3; Smad6 iΔEC/iΔEC , n =3; Alk1 +/iΔEC , n =5; Smad6 iΔEC/iΔEC ; Alk1 +/iΔEC , n =6 livers. One-way ANOVA with Tukey's multiple comparisons test. (D-I) RT-qPCR of PECAM1 + cells from E16.5 livers of Smad6 iΔEC/iΔEC (D-F; WT, n =6; Smad6 iΔEC/iΔEC , n =8 livers) and Smad6 iΔEC/iΔEC ;Alk1 +/iΔEC (G-I; WT, n =7; Smad6 iΔEC/iΔEC ;Alk1 +/iΔEC , n =8 livers) crosses. (D,G) Lyve1, (E,H) stabilin 2 and (F,I) Gata4. CT values normalized to Gapdh and mRNA expression reported as fold change relative to WT average. Data, individual points for each embryo and mean±s.d. Unpaired two-tailed t -test. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001; ns, not significant. Scale bars: 50 µm.

Article Snippet: Livers were rehydrated in a methanol/H 2 O series, rinsed in PBS for 45 min at RT, washed in PTx.2 [0.2% Triton X-100 (Sigma-Aldrich, X100) in PBS] 2× for 30 min at RT, permeabilized in permeabilization solution [0.3 M glycine (Sigma-Aldrich, G7126)+0% DMSO (Fisher D128) in PTx.2] for 1.5 day at 37°C, incubated in blocking solution [6% donkey serum (Sigma-Aldrich, D9663)+10% DMSO in PTx.2] for 1.5 day at 37°C, followed by primary antibody incubation for 3 days at 37°C (Lyve1; R&D Biosystems, AF2126, goat, 0.2 mg/ml stock) diluted 1:100 in PTwH solution [0.2% Tween-20 (Sigma-Aldrich, P9416)+0.01 mg/ml Heparin (Sigma-Aldrich, H3393) in PBS supplemented with 3% donkey serum+5% DMSO].

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Two Tailed Test