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: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

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

Journal: The American Journal of Pathology

Article Title: Characterizing Kupffer Cell Production of CD5 Antigen-Like and Its Function on Regulating Migration of Natural Killer T Cells

doi: 10.1016/j.ajpath.2025.06.003

Figure Lengend Snippet: CD5L promotes the uptake of free fatty acids (FFAs) by hepatocytes and stellate cells. A: Uptake of BODIPY-labeled palmitate acid (BODIPY-PA) in a time-dependent manner. Top four panels: Images taken at indicated time of PA exposure. Bottom panel: Quantitative analysis of the images. B: Cultured mouse hepatocytes ( top panel ) and HepG2 cells ( bottom panel ) were pretreated with CD5L (2000 ng/mL) or vehicle for 25 minutes. Uptake of free fatty acids was then measured by incubating BODIPY-PA for the indicated period. CD5L pretreatment enhanced the uptake of BODIPY-PA with 0.25, 0.5, and 1 hour of uptake but not 2 and 4 hours of uptake. C: Cultured LX-2 cells were pretreated with CD5L (2000 ng/mL) or vehicle for 25 minutes. Uptake of free fatty acids was then measured by incubating BODIPY-PA for the indicated period. CD5L pretreatment facilitated the uptake of BODIPY-PA with 15 minutes, 30 minutes, and 1 hour of uptake but not 2 and 4 hours of uptake. No differences were observed with 4 hours of uptake. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. Scale bars = 50 μm ( A ). NS, not significant.

Article Snippet: Then, cells were treated with rabbit anti-mouse CD5L as primary antibody (ABclonal, Woburn, MA) and BV421 labeled donkey anti-rabbit IgG as the secondary antibody (clone: Poly4064; BioLegend) subsequently.

Techniques: Labeling, Cell Culture

Journal: The American Journal of Pathology

Article Title: Characterizing Kupffer Cell Production of CD5 Antigen-Like and Its Function on Regulating Migration of Natural Killer T Cells

doi: 10.1016/j.ajpath.2025.06.003

Figure Lengend Snippet: Expression of Cd5l in liver tissues from mouse models of steatotic liver injury. A: Expression of Cd5l is concurrently induced with Clec4f , a marker for liver macrophages in high-fat diet (HFD) induced steatotic livers. Low-fat diet (LFD) serves as control. B: Expression of Cd5l and Clec4f in ethanol (EtOH) induced steatotic livers. C : Expression of Cd5l and Clec4f in steatotic livers induced by high-fat and high-sucrose (HFHC) feeding versus normal chow (NC) control. D : Expression of Cd5l in steatohepatitis LiPten livers. E: Correlation of Clec4f and Cd5l expression in all mice analyzed in A – D regardless of diet and genotype. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Con, control.

Article Snippet: Then, cells were treated with rabbit anti-mouse CD5L as primary antibody (ABclonal, Woburn, MA) and BV421 labeled donkey anti-rabbit IgG as the secondary antibody (clone: Poly4064; BioLegend) subsequently.

Techniques: Expressing, Marker, Control

Journal: The American Journal of Pathology

Article Title: Characterizing Kupffer Cell Production of CD5 Antigen-Like and Its Function on Regulating Migration of Natural Killer T Cells

doi: 10.1016/j.ajpath.2025.06.003

Figure Lengend Snippet: Identification of Clec4f + liver macrophages (LMacs) as the dominant cell population expressing CD5L in the liver. A: Analysis of CD5L in all CD45 + cell populations identified a CD5L hi versus a CD5L low population. B: Analysis of CD11b and F4/80 in all CD45 + cell populations to identify CD11b hi F4/80 low (yellow) versus CD11b intermediate F4/80 hi (red) population LMacs. C: Overlay of A and B shows that CD5L hi population overlaps with CD11b intermediate F4/80 hi population LMacs. D: Analysis of CD5L and CLEC4F shows CD5L high expression cells also express high CLEC4F. E: Quantification of CD5L expression in the two LMac populations in LiPten versus control mouse livers. ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001. FSC, forward scatter; MFI, mean fluorescent intensity.

Article Snippet: Then, cells were treated with rabbit anti-mouse CD5L as primary antibody (ABclonal, Woburn, MA) and BV421 labeled donkey anti-rabbit IgG as the secondary antibody (clone: Poly4064; BioLegend) subsequently.

Techniques: Expressing, Control

Journal: The American Journal of Pathology

Article Title: Characterizing Kupffer Cell Production of CD5 Antigen-Like and Its Function on Regulating Migration of Natural Killer T Cells

doi: 10.1016/j.ajpath.2025.06.003

Figure Lengend Snippet: Colocalization of CD5L with CLEC4F in mouse livers. A: Immunofluorescence staining of CD5L and CLEC4F in LiPten versus genotype control (Con) livers showing more CD5L and CLEC4F staining in the injured livers of LiPten mice. Most CD5L is colocalized in cell expression CLEC4F. Blue, DAPI for nuclei. Quantification of positively stained cells. Boxed areas are shown in the bottom three panels as enlarged images. Arrows point to CD5L and CLEC4F positive stained cells. B: Three random sections per animal were counted for double-stained cells versus total CLEC4F staining cells in the LiPten and control mice. The fold change difference between LiPten livers and controls is reported. C: Expression of Clec4f , Cd5l , and TimD4 in livers of mice treated with clodronate (CLD) to depleted macrophages. D: Alamar Blue fluorescent signal in Raw264.7 cells treated with 250 μmol/L palmitic acid with or without CD5L (2000 ng/mL). n = 3 ( B ). ∗ P < 0.05, ∗∗∗∗ P < 0.0001. Scale bars = 100 μm ( A ). PBS, phosphate-buffered saline.

Article Snippet: Then, cells were treated with rabbit anti-mouse CD5L as primary antibody (ABclonal, Woburn, MA) and BV421 labeled donkey anti-rabbit IgG as the secondary antibody (clone: Poly4064; BioLegend) subsequently.

Techniques: Immunofluorescence, Staining, Control, Expressing, Saline

Journal: The American Journal of Pathology

Article Title: Characterizing Kupffer Cell Production of CD5 Antigen-Like and Its Function on Regulating Migration of Natural Killer T Cells

doi: 10.1016/j.ajpath.2025.06.003

Figure Lengend Snippet: CD5L promotes chemotaxis of natural killer T (NKT) cells. A: Schematics of the Transwell assay. B: Transwell assay performed with primary liver immune cells in the insert with or without 2000 ng/mL CD5L in the bottom chamber. No CD5L wells contain media and 0.1% fetal bovine serum (FBS). Cells recovered in the bottom were analyzed for immune cell types using flow cytometry and reported as percentage of cell distributions. C: Transwell assay performed with primary liver immune cells in the insert with or without liver macrophage (LMac) in the bottom chamber. No LMac wells contain media and 0.1% FBS. Cells recovered in the bottom were analyzed for immune cell types using flow cytometry and reported as percentage of cell distributions. Each dot is one mouse. Data are not significant (NS), P = 0.057. D: Transwell assay performed with Jurkat cells in the insert and 2% FBS with indicated doses of CD5L in the bottom chamber. E: Transwell assay performed with primary liver immune cells in the insert and LMac in the bottom chamber. Treatment with or without CD5L neutralizing antibody or CD36 inhibitor SMS121 is compared with vehicle treatment (0.1% FBS). F: Schematic representation of the overall function of CD5L in the liver: CD5L produced by LMacs facilitates lipid uptake in hepatocytes and stellate cells and regulates the migration of NKT cells. G: Correlation of CD5L with Nk1.1, a marker of NKT in liver injury models. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001; # P < 0.05 only detected in experimental group.

Article Snippet: Then, cells were treated with rabbit anti-mouse CD5L as primary antibody (ABclonal, Woburn, MA) and BV421 labeled donkey anti-rabbit IgG as the secondary antibody (clone: Poly4064; BioLegend) subsequently.

Techniques: Chemotaxis Assay, Transwell Assay, Flow Cytometry, Produced, Migration, Marker

Journal: The American Journal of Pathology

Article Title: Characterizing Kupffer Cell Production of CD5 Antigen-Like and Its Function on Regulating Migration of Natural Killer T Cells

doi: 10.1016/j.ajpath.2025.06.003

Figure Lengend Snippet: Elevated plasma CD5L in high-fat diet (HFD)–induced liver injury and its association with hepatocellular carcinoma (HCC) response to immune checkpoint therapy. A: Plasma concentration of CD5L in patients with HCC versus healthy donors. A and B: Plasma concentration of CD5L is higher in patients with HCC with higher Child Pugh scores ( B ) versus lower scores ( A ). C: Plasma concentration of CD5L predicts patient response to therapy. Left panel: Plasma CD5L in patients showed clinical benefit versus those who did not show clinical benefit. Right panel: Plasma CD5L in patients who responded completely or partially versus those whose disease did not progress or progressed. Red dotted line shows a trend of progression. Data are not significant (NS), P = 0.0748. D: Plasma alanine aminotransferase (ALT) levels in mice fed low-fat diet (LFD) versus HFD at the indicated time points. Each dot represents one mouse. E: Detection of CD5L in serum of mice fed LFD versus HFD. HFD feeding for 5 and 9 months significantly increased the plasma levels of CD5l. Each dot represents one mouse. n = 50 patients with HCC ( A ); n = 5 healthy donors ( A ); n = 1 patient who responded completely ( C , right panel ); n = 8 patients who responded partially and patients whose disease progressed ( C , right panel ); n = 27 patients whose disease did not progress ( C , right panel ). ∗ P < 0.05, ∗∗∗ P < 0.001. CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease.

Article Snippet: Then, cells were treated with rabbit anti-mouse CD5L as primary antibody (ABclonal, Woburn, MA) and BV421 labeled donkey anti-rabbit IgG as the secondary antibody (clone: Poly4064; BioLegend) subsequently.

Techniques: Clinical Proteomics, Concentration Assay

Journal: bioRxiv

Article Title: CD5L is a canonical component of circulatory IgM

doi: 10.1101/2023.05.27.542462

Figure Lengend Snippet: A) Representative titration curves for recombinant IgM ± CD5L and serum IgM-CD5L to pIgR, FcαµR and FcµR, of which combined data is shown in . B) Binding of IgM and IgM-Fc (CH2-CH4) ± CD5L to pIgR was also assessed in a reversed setup, where IgM was first captured with an anti-IgM antibody and then binding of biotinylated pIgR was detected with streptavidin-HRP. In both approaches, integration of CD5L reduced binding to the receptor. Data from n=2-3 experiments. Bars and error bars represent mean and S.E. C) Purified serum IgM was reduced using varying concentrations of DTT ranging from 0.06 mM to 1 mM. Reduced IgM was then assessed using HP-SEC and IgM(-CD5L) ELISA. Whereas at high concentrations of DTT IgM falls apart into fragments the size of IgG-like IgM ‘monomers’, at lower concentrations it remains mostly the size consistent with a pentamer. Nevertheless, at these limiting reducing conditions, CD5L is partially released, as assessed by IgM-CD5L complex ELISA (right panel). D) Partial release of CD5L improves binding to pIgR and FcαµR, but not FcµR. Shown is a representative experiment of n=3.

Article Snippet: Cells were stained with FITC-labeled mouse anti-rabbit IgG (2A9, Abcam) and with CD5L-bt followed by streptavidin-APC, after which the double-positive were sorted and seeded in single wells.

Techniques: Titration, Recombinant, Binding Assay, Purification, Enzyme-linked Immunosorbent Assay