Journal: Cancer Communications

Article Title: CD146 promotes malignant progression of breast phyllodes tumor through suppressing DCBLD2 degradation and activating the AKT pathway

doi: 10.1002/cac2.12495

Figure Lengend Snippet: Identified DCBLD2 as an interaction protein to CD146.(A) The workflow for identification of protein complexes and interactions of CD146 in PT cells. Vectors were designed containing transmembrane peptides or CD146 cDNA and C‐terminal tagging with BirA* and flag. The expression vector can then be transfected into malignant PT cells to establish the transgenic stably expressing isogenic cell lines. For the AP‐MS and TurboID analysis approaches, the cell line is separated into 2 cultures, TurboID cells receiving addition of 50 μmol/L biotin in their culture medium. In the following protein extraction process, optimized lysis and affinity purification conditions for both analysis approaches were used. The interacting proteins were then analyzed by quantitative mass spectrometry and high confidence proteins were inferred via stringent statistical filtering.(B) Venn diagram showing the interactors identified in AP‐MS/TurboID assays and the strategy to identify CD146 downstream protein in phyllode tumor.(C) WB showed the changes in the expression level of CD146, DCBLD2, and SEMA4B in benign/malignant cell lines.(D) Immunoprecipitation assay showed the interaction between CD146 and DCBLD2 from SYSH‐MPT‐02. The immunoprecipitants were precipitated using anti‐CD146 mAb AA1 and mIgG as control.(E) Pull‐down assay revealed the direct interaction between CD146 and DCBLD2. Purified His‐tagged DCBLD2 and Fc‐CD146 proteins were incubated in PBS. CD146 and DCBLD2 were pulled down using Protein G PLUS Agarose.(F) Co‐IP assays for association of CD146 mutants with DCBLD2 in HEK293T cells showed CD146 D4‐D5 domain is necessary for binding.(G) Pull‐down assay revealed that anti‐CD146 AA98 antibody blocked the CD146‐DCBLD2 interaction.Abbreviations: CD146, cluster of differentiation 146; DCBLD2, discoidin, CUB And LCCL domain containing 2; Co‐IP, co‐immunoprecipitation; AP‐MS, affinity purification mass spectrometry; HCIP, high‐confidence interaction proteins; PPI, protein‐protein interaction networks; PI3K, phosphatidylinositol‐3‐kinase; AKT, protein kinase B; IP, immunoprecipitation; IB, immunoblotting; SP, signal peptide; Ext, extracellular domain; Tm, transmembrane domain; Cyt, Intracellular domain.

Article Snippet: His‐DCBLD2 and CD146‐Fc or human Fc protein (200 ng/mL; Sino Biological, Beijing, China) were incubated in PBS at 4°C for 1 h. After complete binding, the proteins were immunoprecipitated with Protein G PLUS‐agarose beads (Santa cruz) and the bound proteins were analyzed by WB.

Techniques: Expressing, Plasmid Preparation, Transfection, Transgenic Assay, Stable Transfection, Protein Extraction, Lysis, Affinity Purification, Mass Spectrometry, Immunoprecipitation, Pull Down Assay, Purification, Incubation, Co-Immunoprecipitation Assay, Binding Assay, Western Blot

Journal: Cancer Communications

Article Title: CD146 promotes malignant progression of breast phyllodes tumor through suppressing DCBLD2 degradation and activating the AKT pathway

doi: 10.1002/cac2.12495

Figure Lengend Snippet: CD146 promotes PI3K‐AKT pathway by stabilizing DCBLD2.(A‐B) IB showed the changes in the expression level of DCBLD2 and AKT pathway proteins in CD146‐knock down (A) or DCBLD2‐knock down (B) in SYSH‐MPT‐02 cells.(C) IB showed the changes in the expression level of DCBLD2 and AKT pathway in DCBLD2 rescued KO cell lines.(D) Analysis of DCBLD2 stability with CHX chase assay in control and CD146‐KO SYSH‐MPT‐02 cells. Immunoblot images (upper panel) and quantitative analysis (lower panel) of DCBLD2 in the upper image.(E) Immunoblots with the indicated antibodies on lysates from control and CD146‐KO SYSH‐MPT‐02 treated with MG132. The images and their quantifications are presented in the upper and lower panels, respectively.(F) Co‐IP assays showed the interactions of CD146, DCBLD2, and SEMA4B in HEK293T cells. Proteins were precipitated by indicated anti‐tag mAb and examined by immunoblot.(G) Immunoblots with the ubiquitination antibodies on lysates from transfected HEK293T cell lines treated with MG132.(H) Graphical illustration of the working model.Abbreviations: DCBLD2, discoidin, CUB and LCCL domain containing 2; SEMA4B, semaphorin 4B; Co‐IP, co‐immunoprecipitation; NC, negative control; WT, wild type; KO, knock out; CHX, Cycloheximide, AKT, protein kinase B IP, immunoprecipitation; IB, immunoblotting; Ub, ubiquitination.

Article Snippet: His‐DCBLD2 and CD146‐Fc or human Fc protein (200 ng/mL; Sino Biological, Beijing, China) were incubated in PBS at 4°C for 1 h. After complete binding, the proteins were immunoprecipitated with Protein G PLUS‐agarose beads (Santa cruz) and the bound proteins were analyzed by WB.

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Negative Control, Knock-Out

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Four sutures were placed in the healthy adult mouse cornea on day 0 to induce inflammation. Corneas were harvested on day 12 for immunofluorescence analysis. (B) Whole mount hemisected mouse corneas immunostained for LYVE-1 ( cyan , lymphatic surface marker) and CD31 ( red, pan-endothelial marker). LEC-specific IFT20 KO mice expressed LYVE1Cre and carried two floxed alleles of Ift20 . Littermate controls were negative for Cre and carried one floxed allele of Ift20 . Corneas were imaged using laser scanning confocal microscopy and micrographs shown are maximum intensity projections (MIPs). Scale bar = 500 μm. (C) Quantification of lymphatic vessel area as a percentage of total corneal area from B . (D) Quantification of blood vessel area as a percentage of total corneal area from B . (E) Whole mount immunofluorescence staining for VE-cadherin ( white, VECAD) and LYVE-1 ( blue ) in lymphatic specific-IFT20 KO mice and littermate controls as in B . Corneas were imaged using laser scanning confocal microscopy. Micrographs shown are MIPs from z-stacks encompassing volumes that contain LYVE-1+ lymphatic vessels. Scale bar left = 50 μm. Scale bar right = 25 μm. (F) Quantification of VECAD+ lymphatic vessel (LV) area as a percentage of total LV area. (C, D, F) Each data point represents one field of view. For each quantification, two to four FOVs were quantified from each of four IFT20 KO mice and three negative control littermates. *p<0.05, **p<0.005.

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: Immunofluorescence, Marker, Confocal Microscopy, Staining, Negative Control

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Immunofluorescence micrographs of LYVE-1+ lymphatic vessel morphology in mouse ear skin. LEC-specific IFT20 KO mice expressed LYVE1Cre and carried two floxed alleles of Ift20 . Littermate controls were negative for Cre and carried one floxed allele of Ift20 . Scale bar = 100 μm. (B) To assess the ability of lymphatic vessels to transport lymph, 0.5 μL of TRITC-conjugated dextran (MW 40 kDa) was injected into mouse ear dermis using a stereotactic injector fitted with a pulled glass needle. Fluorescent lymph uptake and transport by lymphatic vessels was assessed by intravital stereofluorescence microscopy. (C-E) Intravital microscopy tracking fluorescent dextran drainage through ear dermis lymphatic vessels in lymphatic-specific IFT20 KO mice and Cre-negative littermate controls. C shows clean transport of TRITC-dextran toward the base of the ear in control mice, while IFT20 KO mice displayed retrograde flow ( red arrows ) and extravascular fluorescent signal in the interstitium. Images shown are from 20 min after injection. (D) Red arrows indicate accumulation of TRITC-dextran in abnormally-shaped lymphatic capillary structures. (E) Red arrows indicate retrograde flow, which is significantly more pronounced in IFT20 KOs. Quantification shows the number of clock hours (from 12 o’clock to 6 o’clock) per injection displaying retrograde flow. Quantification includes data from both 0.5 and 4 μL injections from 3 experiments in IFT20 KO and control mice. p = 0.0001. Scale bars = 100 μm.

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: Immunofluorescence, Injection, Microscopy, Intravital Microscopy, Control

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Ift20 was knocked out of immortalized mouse lymphatic endothelial cells (mLEC) using CRISPR/Cas9. Immunofluorescence micrographs confirm loss of IFT20 ( left panel ) and abrogation of primary cilia assembly in IFT20 KO cells after 24 h serum starvation in media containing 0.2% FBS ( right panel, quantified in B ). DAPI ( blue ). Scale bars = 10 μm. (B) Quantification of primary cilia incidence in parental and IFT20 KO mLECs from A . Data points represent each of three independent experiments, each quantifying 100+ cells. (C) Epifluorescence micrographs of parental and IFT20 KO mLECs with phalloidin-labeled F-actin ( left panel ) or ZO-1 immunofluorescence staining ( right panel ). DAPI ( blue ). Scale bars = 10 μm. (D) Chemotaxis potential of parental and IFT20 KO mLECs was assessed by transwell migration assay. Cells were seeded in the upper chamber in starving media containing 0.2% FBS. Lower wells were filled with media containing either 0.2% FBS, 10% FBS, or 500 ng/mL VEGF-C in 0.2% FBS. After 24 h migration, membranes were fixed, mounted in DAPI mounting media, and imaged via epifluorescence microscopy. Scale bar = 200 μm. (E) Quantification of transwell migration from D . Migration is graphed relative to 10% FBS-stimulated mLEC migration. Each data point represents an average from 5 FOVs per membrane. Quantification is representative of three independent experiments in which three membranes were quantified per experimental condition. (F) Ift20 was targeted by siRNA in primary human dermal lymphatic endothelial cells (HDLECs). F-actin was labelled with phalloidin, and ZO-1 and VE-cadherin were labeled by immunofluorescence. Micrographs are MIPs from laser scanning confocal microscopy. DAPI ( blue ). Scale bars F-actin and ZO-1 = 10 μm. Scale bar VECAD left panel = 50 μm, right panel = 10 μm. *p<0.05, **p<0.005.

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: CRISPR, Immunofluorescence, Labeling, Staining, Chemotaxis Assay, Transwell Migration Assay, Migration, Epifluorescence Microscopy, Membrane, Confocal Microscopy

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Control and IFT20 KD HDLECs in homeostasis were immunostained for IFT20 and RAB5. Micrographs are MIPs from laser scanning confocal microscopy. Scale bars = 25 μm. (B) Quantification of integrated intensity from RAB5+ area in control and IFT20 KD HDLECs. Intensities are graphed relative to average control HDLEC RAB5 intensity. Quantification is from one of three biological replicates and is representative of 300+ control and 300+ IFT20 KD HDLECs. (C) Control and IFT20 KD HDLECs were serum starved in media containing 1/5 EGM-MV2 and 4/5 EBM-2 (hereafter, starving media) for 24 h. Cells were then either: placed in fresh starving media for 45 min, then fixed; placed in fresh starving media + 2 μg/mL VEGF-C for 45 min, then fixed; or placed in fresh starving media + 2 μg/mL VEGF-C for 45 min, then placed in EBM-2 basal media for a 90 min washout, then fixed. RAB5 was detected in fixed cells by immunofluorescence. Micrographs are MIPs from laser scanning confocal microscopy. Scale bar = 25 μm. (D) Quantification of C . Integrated intensities from RAB5+ area are graphed for control and IFT20 KD HDLECs separately and combined. Intensities are graphed relative to average control HDLEC RAB5 intensity in the 45 min starve condition. Data points represent the average of three FOVs within one technical replicate. Each of three independent experiments included two or three technical replicates (three FOVs each) per experimental condition. Quantification is representative of 300+ control and 300+ IFT20 KD HDLECs. *p<0.05, **p<0.005, ***p<0.001.

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: Control, Confocal Microscopy, Immunofluorescence

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Control and IFT20 KD HDLECs were serum starved for 24 h in 1/5 EGM-2MV and 4/5 EBM-2 (hereafter, starving media). Cells were then treated with 1 μg/mL VEGF-C for 15 or 30 min in starving media and lysed or placed in EBM-2 basal media for 30 min washout. Lysates were subjected to SDS-PAGE and western blot for the indicated proteins. β-actin is shown as a loading control below each band, respectively. β-actin shown in the 7 th row applies to both pVECAD and pERK1/2. (B) Protein levels were quantified by densitometry including background subtraction locally around each band and normalized to β-actin and, where indicated, levels of total protein (tERK1/2, tAKT). Each data point represents a single densitometry value from a total of four biological replicates. Data are graphed relative to column 2 (control cells stimulated with VEGF-C for 15 min) set equal to 1. (C) Control and IFT20 KD HDLECs in homeostasis were immunostained for PROX1. Scale bar = 25 μm. Quantification of integrated intensity from PROX1+ area in control and IFT20 KD HDLECs. Intensities are graphed relative to average control HDLEC PROX1 intensity. Data points represent the average from three FOVs in one technical replicate. Each of three independent experiments included two or three technical replicates (three FOVs each) per experimental condition. Quantification is representative of 300+ control and 300+ IFT20 KD HDLECs. *p<0.05, **p<0.005, ***p<0.001.

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: Control, SDS Page, Western Blot

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Non-serum starved control HDLECs were immunostained for IFT20 ( green ) and RAB5 ( magenta ). Scale bars = 10 μm. (B) Control HDLECs were serum starved for 24 h in 1/5 EGM-2MV and 4/5 EBM-2 (hereafter, starving media) and then treated for 6 h with 2 μg/mL VEGF-C in starving media. Cells were then fixed or placed into EBM-2 basal media for 1.5 or 3 h washout. IFT20 ( green ) and RAB5 ( magenta ) were detected by immunofluorescence microscopy. (A-B) Micrographs are single 0.3 μm z-slices from laser scanning confocal microscopy. DAPI ( blue ). Scale bars = 10 μm. (C) Quantification of B . RAB5+ IFT20+ area is graphed as a percentage of total IFT20+ area per FOV representing one of two biological replicates. Quantification is representative of 100+ control and 100+ IFT20 KD HDLECs. ***p<0.001, ****p<0.0001.

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: Control, Immunofluorescence, Microscopy, Confocal Microscopy

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: (A) Control and IFT20 KD HDLECs were serum starved for 24 h in in 1/5 EGM-2MV and 4/5 EBM-2 (hereafter, starving media). Cells were then treated with 2 μg/mL VEGF-C for 6 h in starving media and fixed or placed in EBM-2 basal media for 1.5 or 3 h washout. VE-cadherin ( white ) was detected by immunofluorescence microscopy. Scale bar = 50 μm. (B) Quantification of granularity to measure breakdown in VE-cadherin cell-cell junctions from A . Each data point represents the average granularity of a single FOV normalized per cell from each of two biological replicates. *p<0.05, **p<0.005. (C) Control and IFT20 KD HDLECs were serum starved for 24 h in starving media. Cells were then treated with 2 μg/mL VEGF-C for 6 h in starving media and fixed or placed in EBM-2 basal media for 1.5 or 3 h washout. VE-cadherin ( green ) and RAB5 ( magenta ) were detected by immunofluorescence microscopy. Scale bar = 10 μm. (D) Quantification of VE-cadherin+ RAB5+ area from C . Graphs show one representative biological replicate of two, each comprising two technical replicates with 100+ cells per condition. In graphs depicting individual CTRL or KD data, data is graphed relative to control 24 h starve treatment (column 1). In the right graph where the two cell lines are combined, data is graphed as total area (px 2 ) for both datasets. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. (A, C) Micrographs are single 0.3 μm z-slices from laser scanning confocal microscopy. DAPI ( blue ).

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: Control, Immunofluorescence, Microscopy, Confocal Microscopy

Journal: bioRxiv

Article Title: IFT20 regulates lymphatic endothelial cell-cell junctions via endocytic trafficking of VE-cadherin

doi: 10.1101/2025.01.15.631989

Figure Lengend Snippet: Model: In the absence of IFT20, VE-cadherin accumulates in RAB5+ endosomes. This results in discontinuous cell-cell junctions and increased lymphatic vessel permeability. Sequestration of VE-cadherin inside the cell enables internalization and sustained signaling of VEGFR-3, promoting lymphangiogenesis. Model created with BioRender.com .

Article Snippet: Primary antibodies utilized in cell and tissue immunofluorescence staining include: rabbit anti-mouse LYVE-1 1:200 (abcam, ab33682), rat anti-mouse LYVE-1 1:50 (Santa Cruz, sc-65647), goat anti-human PROX-1 1:200 (R&D Systems, AF2727), rabbit anti-mouse PROX-1 1:500 (abcam, ab101851), mouse anti-mouse ARL13B 1:200 and 1:4,000 (Neuromab, 75-287), rabbit anti-mouse IFT20 1:1,000 (Proteintech, 13615-1-AP), rabbit anti-mouse ZO-1 1:1,000 (Proteintech, 21773-1-AP), goat anti-mouse VE-cadherin 1:200 (R&D systems, AF1002), rat anti-mouse VE-cadherin 1:1,000 (BD Bioscience, 555289), and rat anti-PECAM-1 1:1,000 (BD Pharmingen, 553370).

Techniques: Permeability