Journal: Cell Death Discovery

Article Title: CD5L deficiency attenuate acetaminophen-induced liver damage in mice via regulation of JNK and ERK signaling pathway

doi: 10.1038/s41420-021-00742-3

Figure Lengend Snippet: APAP was intraperitoneally injected into wild-type (WT) mice ( n = 5 for each time point) at 300 mg/kg, and the CD5L level in the liver at specified time point was detected by A q-PCR and B western blotting; C Immunohistochemical staining of CD5L in liver sections of WT ( n = 4 for each group) treated with PBS or APAP for 24 h, representative images are shown; D The expression of CD5L in human liver tissue ( www.proteinatlas.org ); E The diagram of CRISP/Cas9 CD5L knockout strategy; F The expression of CD5L protein in the liver of WT and CD5L -/- mice was detected by western blotting; G Immunohistochemical staining of CD5L in liver sections of WT or CD5L −/− mice ( n = 4 for each group), representative images are shown; H , I Serum ALT and liver H&E staining at 8 h and 24 h after PBS or APAP injection in mice ( n = 10 for each group). Necrotic area was measured by Image J. Representative images are shown. Scale: 100 μm. The results are presented as means ± SD of at least three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Immunohistochemical (IHC) staining was performed using CD5L antibody (#50020-T24, Sino Biological, Beijing, China), PCNA antibody (#13110, Cell Signaling Technology, Beverly, MA, USA) and sheep anti-APAP polyclonal antibody (#0016-0104, Bio-Rad, Düsseldorf, Germany).

Techniques: Injection, Western Blot, Immunohistochemical staining, Staining, Expressing, Knock-Out

Journal: Cell Death Discovery

Article Title: CD5L deficiency attenuate acetaminophen-induced liver damage in mice via regulation of JNK and ERK signaling pathway

doi: 10.1038/s41420-021-00742-3

Figure Lengend Snippet: A Immunohistochemical staining of PCNA in WT and CD5L −/− mouse liver at the indicated time point after PBS or APAP treatment ( n = 5 for each group at each time point). Deeply stained nucleus indicates positive results. The ratio of PCNA positive cells to total hepatocytes was calculated. Representative images are shown. Scale: 100 μm. B Vascular endothelial permeability after APAP treatment for 24 h was determined by Evans Blue assay ( n = 8 for each group). The results are presented as means ± SD of at least three independent experiments. * P < .05; ** P < 0.01.

Article Snippet: Immunohistochemical (IHC) staining was performed using CD5L antibody (#50020-T24, Sino Biological, Beijing, China), PCNA antibody (#13110, Cell Signaling Technology, Beverly, MA, USA) and sheep anti-APAP polyclonal antibody (#0016-0104, Bio-Rad, Düsseldorf, Germany).

Techniques: Immunohistochemical staining, Staining, Permeability, Evans Blue Assay

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: Serum proteins separated by two dimensional gel electrophoresis in early and advanced NAFLD, with and without HCC . These are representative blots comparing immune depleted and desalted serum from a patient with NAFLD and no fibrosis, a patient with NAFLD cirrhosis, and a patient with NAFLD cirrhosis and cancer. Serum profiles separated by 2D gel electrophoresis from 5 patients in each group, run in duplicate, were compared using Progenesis software. 5 spots were characterised by MS after excision. Spots 1,2 and 3 were isoforms of apolipoprotein A1, spot 4 was apolipoprotein A4, and spot 5 was identified as CD5 antigen like, or CD5L.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Software

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates poorly between cirrhotic patients with and without HCC . Serum levels of CD5L were measured by ELISA assay in cirrhotic patients with (218 ± 221; n = 45) and without (194 ± 166; n = 49) HCC, as shown in 2A. Characteristics of the individuals are shown in Table 1. There was no significant difference between the two groups. ROC analyses is shown in 2B. The area under the curve is 0.495 (95% confidence intervals 0.376 and 0.614). A level > 400 ng/ml has a sensitivity of only 20%, but a specificity of 88%. Levels > 500 ng/ml have a specificity of 96%, > 600 ng/ml of 98% and > 700 ng/ml of 100%. For comparison, the mean CD5L serum level from patients without cirrhosis (detailed in figure 3) is also shown.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates between steatohepatitis patients with and without cirrhosis . Serum levels of CD5L were measured in a total of 113 patients with either ALD or NAFLD. Fibrosis was scored histologically on liver biopsy as per the Brunt scoring system. The numbers of individuals in each group are shown within the boxes of chart 3A. The difference in CD5L levels between the different stages of fibrosis is statistically significant by univariate analysis (p = 0.004) controlled for both age and sex. This difference The ROC analyses for the identification of those individuals with stage 4 fibrosis (cirrhosis) is depicted in 3B. The area under the curve is 0.719 (95% confidence intervals 0.623 and 0.816), p < 0.0001. A level of CD5L 50 ng/ml has a sensitivity of 78% and a specificity of 46%, while a level of 100 ng/ml has a sensitivity of 63% and a specificity of 72%. A level greater than 200 ng/ml has a specificity of 95% (sensitivity 41%), and greater than 300 ng/ml of 97% (sensitivity 27%).

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques:

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L mRNA expression is not altered in association with either fat, inflammation or fibrosis scores in pre-cirrhotic NAFLD liver tissues . mRNA CD5L expression was quantified by real-time PCR, relative to GAPDH and a normal liver sample, in 21 pre-cirrhotic NAFLD biopsy tissues, 13 normal liver samples taken at the time of liver resection, one cirrhotic liver and one HCC. As shown in 4A-C, there was no difference in any pre-cirrhotic NAFLD biopsy tissues in association with the degree of fat, inflammation or fibrosis. There was a significant increase in the NAFLD tissues as a group (n = 21), compared with normal liver tissues (n = 13) as represented in 4D (6.945 ± 0.722 versus1.68 ± 0.269; p = 0.000, ***). The elevated CD5L mRNA expression in one cirrhotic and HCC tissue pair, obtained at the time of laparoscopic radiofrequency ablation, is also presented in 4C and is in keeping with the elevated serum CD5L levels identified in the larger cohort of patients studied.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: The identification of the differentiating protein spots on 2D gels.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Sequencing

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: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: Characterization of myeloid cell subsets and identification of CD5L⁺ macrophages as potential migrasome-responsive cells. ( A ) UMAP visualization showing the reclustering of myeloid cells into 10 distinct subpopulations. ( B ) Heatmap displaying the expression profiles of representative marker genes for each myeloid subset. ( C ) Bar plot showing the relative abundance of each myeloid subpopulation in normoxic versus hypoxic migrasome-treated groups. A notable 8-fold increase in the proportion of CD5L⁺ macrophages was observed in the hypoxic group. ( D ) Gene Set Variation Analysis (GSVA) indicating significant activation of the complement signaling pathway in CD5L⁺ macrophages. ( E ) Chord diagram depicting inferred cell–cell communication networks, highlighting potential interactions between CD5L⁺ macrophages and fibroblasts, T cells, and other immune cells via GDF signaling. ( F ) Activation IGF signaling pathway network mediated crosstalk between CD5L⁺ macrophages and epithelial tumor cells. ( G ) In the IGF signaling pathway network, CD5L⁺ macrophages exhibit specific overexpression of IGF1. ( H ) Among the six macrophage cell-types, CD5L⁺ macrophages exhibit specific overexpression of CD5L

Article Snippet: The pellet was resuspended in 200 μL antibody dilution buffer (2% FBS in 1× PBS) and incubated with 0.5 μg CD5L antibody (BAF2834, R&D Systems) at 4 °C for 60 min in the dark.

Techniques: Expressing, Marker, Activation Assay, Over Expression

Journal: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: Phagocytic activity and immunofluorescence validation of CD5L⁺ macrophages in CRC liver metastases following migrasome treatment. ( A ) UMAP blot showing the expression of migrasome marker TSPAN4 in myeloid subsets. ( B ) Boxplot showing efferocytosis scores across the 10 identified myeloid cell subtypes. ( C ) Violin plots depicting the expression of efferocytosis markers CD300B, MERTK, and CD300D across 10 distinct myeloid cell subtypes. ( D ) UMAP plots displaying the expression patterns of three efferocytosis-associated marker genes specifically enriched in CD5L⁺ macrophages. ( E ) Immunofluorescence staining of tumor tissues from MC38-tumor bearing mice showing colocalization of CD163, CD5L, and the migrasome marker MERTK in both treatment groups. Increased MERTK expression is observed in the hypoxic group, indicating enhanced migrasome targeting of CD5L⁺ macrophages

Article Snippet: The pellet was resuspended in 200 μL antibody dilution buffer (2% FBS in 1× PBS) and incubated with 0.5 μg CD5L antibody (BAF2834, R&D Systems) at 4 °C for 60 min in the dark.

Techniques: Activity Assay, Immunofluorescence, Biomarker Discovery, Expressing, Marker, Staining

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD4 + T cells secret p19 after activation through TCR ligation and co-stimulatory signal via CD28 enhances it. ( a ) Mouse T cell hybridomas (DO.BW, 2HCa2, 530, 447, KE8, 216, 118, 1D1-E7, 2B4) and lymphoma (DO11.10) were stimulated with plate-bound anti-CD3 (2 μg/ml ) and anti-CD28 (1 μg/ml ) for 48 h, and culture supernatants were collected and analyzed for p19 by ELISA. ( b – d ) 2B4 cells were stimulated with plate-bound anti-CD3 in the presence or absence of anti-CD28 for indicated time points, and total RNA was extracted and analyzed for mRNA expression by semiquantitative RT-PCR ( b ). PEC macrophages prepared from mice 4 days after the injection with thioglycolate were stimulated with LPS (100 ng/ml) and IFN-γ (1000 U/ml) for 24 h and used as a positive control. Forty-eight hours later, culture supernatants were collected, cell lysates were prepared, and both were analyzed for protein expression of p19 or EBI3 by western blotting ( c ). Culture supernatants from HEK293T cells transfected with expression vectors of pCXN2-p19 or pCXN2-EBI3 were used as positive controls. Culture supernatants of the activated 2B4 cells were also analyzed for p40 together with p19 via ELISA. Culture supernatants of LPS-stimulated splenocytes were used as the positive control ( d ). ( e ) Naive CD4 + T cells from WT mice were stimulated with plate-bound anti-CD3 and anti-CD28 for indicated time points, and total RNA was extracted and analyzed for mRNA expression by semiquantitative RT-PCR. Mouse macrophage cell line, J774.1, and PEC macrophages stimulated with LPS and IFN-γ were used as positive controls. ( f , g) Naive CD4 + T cells were stimulated with plate-bound anti-CD3 (0, 0.2, 0.6 and 2 μg/ml) in the presence or absence of anti-CD28 (1 μg/ml) for 48 h, and total RNA was extracted and analyzed for mRNA expression by semiquantitative RT-PCR ( f ). Culture supernatants of similarly activated naive CD4 + T cells with plate-bound anti-CD3 (0, 0.5, 1 and 2 μg/ml) were collected at the indicated time points and analyzed for IL-2 or p19 by ELISA ( g ). Data are representative of more than two independent experiments.

Article Snippet: The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions.

Techniques: Activation Assay, Ligation, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Positive Control, Western Blot, Transfection

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD4 + T cell-specific conditional p19-deficient mice show alleviated EAE with reduced frequency of GM-CSF + CD4 + T cells in the CNS. Control p19 flox/flox mice or CD4 + T-cell-specific conditional 19-deficient (CD4-Cre/p19 flox/flox ) mice were immunized with MOG 35-55 peptide and their clinical scores were monitored with time ( a ). On day 14, spinal cords and brains were harvested, and the CNS was histopathologically analyzed with H&E staining. Representative images are shown ( b ). On day 41, mononuclear cells were isolated from the CNS, and intracellular cytokine staining was performed after restimulation with PMA and ionomycin. Representative dot plots of GM-CSF and IL-17A in CD4 + T cells are shown ( c ). Average frequencies of respective CD4 + T cells were calculated and compared ( d ). Response to the recall antigen MOG 35-55 peptide was examined using spleen cells ( e ) and mononuclear cells infiltrating the CNS ( f ) on day 14. The culture supernatants were analyzed via ELISA for cytokine production as indicated. Data are shown as mean ± SD (n = 3–7) and are representative of two independent experiments. P values were determined using unpaired, two-tailed Student’s t -test. * P < 0.05, ** P < 0.01.

Article Snippet: The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions.

Techniques: Control, Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: Differentiation into GM-CSF-producing CD4 + T cells is impaired in p19-deficient CD4 + T cells in vitro. Naive CD4 + T cells from WT mice or complete p19-deficient mice (p19 KO) were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) for 4 days under various Th-polarizing conditions; Th, Th0, Th1, ThGM, nonpathogenic Th17, and pathogenic Th17. These cells were then restimulated with PMA and ionomycin, and the intracellular cytokine staining was performed. Representative dot plots for GM-CSF, IL-17A, IFN-γ, and IL-10 in CD4 + T cells are shown ( a ), and average frequencies of respective CD4 + T cells were calculated and compared ( b ). Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test. * P < 0.05, *** P < 0.001.

Article Snippet: The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions.

Techniques: In Vitro, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19 associates with CD5L to form a putative heterodimer of p19/CD5L. ( a ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC and cultured for 3 days, and total cell lysates or culture supernatants were immunoprecipitated with anti-FLAG or anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-c-MYC or biotin-conjugated anti-p19, respectively. Immunoprecipitated protein was confirmed by western blotting with the antibody used for immunoprecipitation. ( b ) HEK293T cells were transiently transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, or both and p3 × FLAG-CMV-14-Hyper-p19/CD5L, then cultured for 3 days. The culture supernatants were then subjected to p19/CD5L-specific ELISA in triplicate. Data are shown as the mean ± SD. ( c ) Naive CD4 + T cells were stimulated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) under pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 8 times, and subjected to immunoprecipitation with anti-CD5L followed by western blotting with biotin-conjugated anti-p19 and subsequently anti-CD5L. ( d ) Naive CD4 + T cells were also similarly stimulated with plate-coated anti-CD3 and anti-CD28 under Th, Th0 and pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 10 times, followed by p19/CD5L-specific ELISA in triplicate. ( e ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 WT or p3 × FLAG-CMV-14-p19 C55S and pCMV3-CD5L-c-MYC and cultured for 3 days. The culture supernatants were immunoprecipitated with anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-p19, then anti-c-MYC. Data are shown as the mean ± SD and representative of four ( a ) or two ( b – e ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05.

Article Snippet: The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions.

Techniques: Cell Culture, Immunoprecipitation, Control, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Centrifugation

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD5L-deficient mice show alleviated EAE with reduced frequency of GM-CSF + CD4 + T cells in the CNS. ( a – d ) WT mice or CD5L-deficient mice were immunized with MOG 35-55 peptide and their clinical scores were monitored with time ( a ). On day 15, spinal cords and brains were harvested, and the CNS was histopathologically analyzed with H&E staining. Representative images are shown ( b ). Mononuclear cells were also isolated from the CNS, and intracellular cytokine staining was performed after restimulation with PMA and ionomycin. Representative dot plots of GM-CSF, and IL-17A in CD4 + T cells are shown ( c ). Average frequencies of respective CD4 + T cells were calculated and compared ( d ). Blood was also taken over time and serum levels of p19/CD5L and CD5L were determined by ELISA ( e , f ). Data are shown as mean ± SD (n = 4–6) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test ( a , d ) or one-way ANOVA ( e , f ). * P < 0.05, ** P < 0.01, *** P < 0.001. NS, not significant.

Article Snippet: The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions.

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone efficiently induces cell proliferation, STAT5 phosphorylation. ( a , b ) HEK293T cells were transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, p3 × FLAG-CMV-14-hyper-p19/CD5L, and p3 × FLAG-CMV-13-hyper-p40/CD5L. The cells were also cotransfected with expression vectors of p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC, together with those of p3 × FLAG-CMV-14-p19 and p3 × FLAG-CMV-14-p40, which produced IL-23 as the positive control. The total amount of DNA in each transfection sample was adjusted to be kept equal with the empty vector. Three days later, culture supernatants were collected, and 10% or 30% of them were used for stimulation of Ba/F3 cells expressing gp130/IL-12Rβ1/IL-12Rβ2/IL-23Rα. For p19 + p40 (IL-23), only 10% culture supernatant was added; therefore, “ – ” means that no data exist for the 30% lane ( a ). Ba/F3 cells were also stimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L (all 20 ng/ml), and hyper-p19/CD5L (0.1 – 20 ng/ml) ( b ). Proliferative activity of these cells was determined by measuring 3 H-thymidine incorporated into the DNA. ( c , d ) Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml ) and anti-CD28 (1 μg/ml ) for 3 days under Th conditions, washed, and rested in 10% FBS medium for 6 h. These cells were unrestimulated ( – ) or then restimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L, hyper-p19/CD5L, IL-27 (positive control for phosphorylation of STAT1 and STAT3, all 20 ng/ml), or IL-2 (positive control for phosphorylation of STAT5, 100 U/ml) for 5, 15 and 60 min, and subjected to western blotting with anti-pY-STATs and subsequently anti-total STATs ( c ). FACS analysis was also performed using anti-pY-STAT5 and its control antibody after stimulation for 20 min ( d ). Data are shown as the mean ± SD in triplicate and are representative of three ( a , c , d ) or two ( b ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions.

Techniques: Phospho-proteomics, Transfection, Control, Plasmid Preparation, Expressing, Produced, Positive Control, Purification, Recombinant, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone induces augmentation of GM-CSF expression. Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) in the presence of recombinant p19, CD5L (all 20 ng/ml), and hyper-p19/CD5L (2–20 ng/ml) under Th conditions for 3 days, and subjected to intracellular staining of GM-CSF and IFN-γ ( a , b ). Representative dot plots were shown ( a ). Culture supernatants were analyzed for GM-CSF by ELISA ( c ). Similarly, naive CD4 + T cells from CD5L-deficient mice were stimulated and subjected to intracellular staining of GM-CSF and IFN-γ ( d , e ). Representative dot plots were shown ( d ). Culture supernatants were analyzed for GM-CSF by ELISA ( f ). Data are shown as mean ± SD in triplicates and are representative of more than two independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions.

Techniques: Expressing, Recombinant, Staining, Enzyme-linked Immunosorbent Assay