Journal: International immunopharmacology

Article Title: Minnelide combined with Angptl3 knockout completely protects mice with adriamycin nephropathy via suppression of TGF-β1-Smad2 and p53 pathways.

doi: 10.1016/j.intimp.2022.109656

Figure Lengend Snippet: Fig. 2. Minnelide alleviates podocyte injury in Angptl3 knockout mice with adriamycin nephropathy. (A) Immunofluorescence of nephrin, podocin, and cd2ap in kidney tissues of three groups of mice. (B and C) The protein expression levels of nephrin, podocin, and cd2ap in kidney tissues of three groups of mice were measured by western blot. n = 3, ****P < 0.0001 vs Angptl3-/- group; #P < 0.05, ##P < 0.01 and ####P < 0.0001 vs ADR + Angptl3-/- group.

Article Snippet: The membrane was incubated overnight at 4 ◦C with the following primary antibodies: nephrin (1:500, Invitrogen, PA5-106921), podocin (1:500, Proteintech, 20384–1-AP), cd2ap (1:500, Invitrogen, PA5-51894), α-SMA (1:1000, Servicebio, GB111364), TGF-β1 (1:500, Servicebio, GB111876), pSmad2 (1:500, Servicebio, GB11343), pp53 (1:500, Servicebio, B. Ji et al. International Immunopharmacology 115 (2023) 109656 GB114061), p53 (1:500, Proteintech, 10442–1-AP), Bax (1:500, Abcam, ab216494), Bcl-2 (1:500, Abcam, ab196495), and Cleaved-caspase3 (1:500, Servicebio, GB11532).

Techniques: Knock-Out, Immunofluorescence, Expressing, Western Blot

Journal: International immunopharmacology

Article Title: Minnelide combined with Angptl3 knockout completely protects mice with adriamycin nephropathy via suppression of TGF-β1-Smad2 and p53 pathways.

doi: 10.1016/j.intimp.2022.109656

Figure Lengend Snippet: Fig. 8. Triptolide attenuates podocyte injury in Angptl3 knockout primary podocytes. (A) Immunofluorescence of nephrin, podocin, and cd2ap in Angptl3 knockout primary podocytes. (B) The expression of podocin and cd2ap proteins in Angptl3 knockout primary podocytes was measured by western blot. n = 3. ****P < 0.0001 vs Angptl3-/- group; ##P < 0.01 and ####P < 0.0001 vs ADR + Angptl3-/- group.

Article Snippet: The membrane was incubated overnight at 4 ◦C with the following primary antibodies: nephrin (1:500, Invitrogen, PA5-106921), podocin (1:500, Proteintech, 20384–1-AP), cd2ap (1:500, Invitrogen, PA5-51894), α-SMA (1:1000, Servicebio, GB111364), TGF-β1 (1:500, Servicebio, GB111876), pSmad2 (1:500, Servicebio, GB11343), pp53 (1:500, Servicebio, B. Ji et al. International Immunopharmacology 115 (2023) 109656 GB114061), p53 (1:500, Proteintech, 10442–1-AP), Bax (1:500, Abcam, ab216494), Bcl-2 (1:500, Abcam, ab196495), and Cleaved-caspase3 (1:500, Servicebio, GB11532).

Techniques: Knock-Out, Immunofluorescence, Expressing, Western Blot

Journal: Microorganisms

Article Title: SLAMF7/STAT6 Pathway Inhibits Innate Immune Response in Late-Stage Human Acanthamoeba Keratitis: A Comparative Transcriptome Analysis.

doi: 10.3390/microorganisms11020365

Figure Lengend Snippet: Figure 5. Representative images of CD8a, CD4, SLAMF7, and STAT6 immunohistochemistry staining in the different groups. Scale bars: 100 µm.

Article Snippet: In addition to the anti-human SLAMF7 and STAT6 antibodies described above, the rabbit anti-human phospho-stat6 antibody (1:1000; Cell Signaling Technology, #9361) and GAPDH antibody (1:2000; Proteintech, Rosemont, IL, USA, #1E6D9) were also used for Western analysis.

Techniques: Immunohistochemistry, Staining

Journal: Microorganisms

Article Title: SLAMF7/STAT6 Pathway Inhibits Innate Immune Response in Late-Stage Human Acanthamoeba Keratitis: A Comparative Transcriptome Analysis.

doi: 10.3390/microorganisms11020365

Figure Lengend Snippet: Figure 6. RT-qPCR and Western blot analysis confirming the validity of the RNA-seq results. (A) The relative expression levels of the DEGs between the AK and control groups were analyzed by RT-qPCR. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) The protein levels of SLAMF7, pSTAT6, and STAT6 in cornea. (C) Correlation of the gene expression levels between RT-qPCR and bioinformatics data.

Article Snippet: In addition to the anti-human SLAMF7 and STAT6 antibodies described above, the rabbit anti-human phospho-stat6 antibody (1:1000; Cell Signaling Technology, #9361) and GAPDH antibody (1:2000; Proteintech, Rosemont, IL, USA, #1E6D9) were also used for Western analysis.

Techniques: Quantitative RT-PCR, Western Blot, RNA Sequencing, Expressing, Control, Gene Expression

Journal: Frontiers in Pharmacology

Article Title: Shensu IV maintains the integrity of the glomerular filtration barrier and exerts renal protective effects by regulating endogenous hydrogen sulfide levels

doi: 10.3389/fphar.2024.1447249

Figure Lengend Snippet: FIGURE 6 Shensu IV regulates the PI3K/AKT signaling pathway through H2S. (A) The effects of Shensu IV and NaHS on the mRNA expression of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, and AKT in renal tissue of PAN rats were analyzed by RT-qPCR. (B) Western blot analysis of the effects of Shensu IV and NaHS on the protein levels of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, p-PI3K,AKT,p-AKT in renal tissue of PAN rats. *P< 0.05, **P< 0.01, ***P< 0.001. Abbreviations: CD2AP, CD2-associated protein; CBS, Cystathionine β-synthase; CSE, Cystathionine γ-lyase; PI3K, Phosphoinositide 3-Kinase; AKT, Protein Kinase B.

Article Snippet: Membranes were blocked with 5% skim milk (Solarbio) to prevent nonspecific binding and then incubated with primary antibodies against CD2AP (1:2000; A01756-2, BOSTER, Wuhan, China), nephrin (1:2000; A01756-2, BOSTER), CBS (1: 10,000; 14787-1-AP, Proteintech, Wuhan, China), CSE (1: 4,000; 12217-1-AP, Proteintech), PI3K (1:2000; 60225-1-Ig, Proteintech), p-PI3K (1:2000; bs-3332R, BOSTER), AKT (1:10,000; 60203-2-Ig, Proteintech), p-AKT (1: 10,000; 66,444-1-lg, Proteintech), NOX4 (1: 8,000; 14347-1-AP, Proteintech), and GAPDH (1:40,000; 60004-1- Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Shensu IV maintains the integrity of the glomerular filtration barrier and exerts renal protective effects by regulating endogenous hydrogen sulfide levels

doi: 10.3389/fphar.2024.1447249

Figure Lengend Snippet: FIGURE 8 Shensu IV regulates the PI3K/AKT signaling pathway through H2S in podocytes. (A) The effects of Shensu IV and NaHS on the mRNA expression of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, and AKT in podocytes were analyzed by RT-qPCR. (B) Western blot analysis of the effects of Shensu IV and NaHS on the protein levels of CD2AP, nephrin, CBS, CSE, NOX4, PI3K, p-PI3K,AKT,p-AKT in PAN-induced podocyocytes. *P< 0.05, **P< 0.01, ***P< 0.001. Abbreviations: CD2AP, CD2-associated protein; CBS, Cystathionine β-synthase; CSE, Cystathionine γ-lyase; PI3K, Phosphoinositide 3-Kinase; AKT, Protein Kinase B.

Article Snippet: Membranes were blocked with 5% skim milk (Solarbio) to prevent nonspecific binding and then incubated with primary antibodies against CD2AP (1:2000; A01756-2, BOSTER, Wuhan, China), nephrin (1:2000; A01756-2, BOSTER), CBS (1: 10,000; 14787-1-AP, Proteintech, Wuhan, China), CSE (1: 4,000; 12217-1-AP, Proteintech), PI3K (1:2000; 60225-1-Ig, Proteintech), p-PI3K (1:2000; bs-3332R, BOSTER), AKT (1:10,000; 60203-2-Ig, Proteintech), p-AKT (1: 10,000; 66,444-1-lg, Proteintech), NOX4 (1: 8,000; 14347-1-AP, Proteintech), and GAPDH (1:40,000; 60004-1- Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: CD2 costimulation strength: A key regulator of T cell function and anti-tumor immunity that is epigenetically regulated

doi: 10.1016/j.isci.2025.113977

Figure Lengend Snippet: A CD2 low phenotype exhibited by brain tumor-infiltrating CD8 + and CD4 + T cells (A and B) (left) , Representative flow cytometry histogram of CD2 expression in naive (blue), central memory (CM-green), effector memory (EM-mangenta), and effector-memory re-expressing CD45RA (EMRA-grey) CD8 + and CD4 + T cells, respectively, from peripheral blood (PB-light color histograms) and tumor (solid color histograms) from one glioblastoma patient. (A and B), (right) , Relative CD2 expression in T cell subsets, normalized to CD2 expression in PB naive CD4 + T cells, per donor per tissue as in (A and B, left) across 5 patients; open-light color circles represent PB T cell subsets, solid rhombus represent core side of tumor, and solid square peripheral side of tumor T cells. (C and D) Same as in (A and B) but for 11 patients with meningioma. Also see and .

Article Snippet: CD2 (3A10B2) Mouse , Proteintech , Cat# 60005-1-Ig.

Techniques: Flow Cytometry, Expressing

Journal: iScience

Article Title: CD2 costimulation strength: A key regulator of T cell function and anti-tumor immunity that is epigenetically regulated

doi: 10.1016/j.isci.2025.113977

Figure Lengend Snippet: Genome-scale CRISPR-CAS9 screen identifies regulators of CD2 expression (A) Diagram shows the experimental design used for identifying positive regulators of CD2 using a GW CRISPR-CAS9 KO screen in the human Jurkat T cell line. CD2lo expressing cells were FACsorted (step 3) at 5 separate timepoints during the cell library screen culture. (B) A representative example plot from one of the five timepoints, showing the distribution of target gene enrichment within the CD2lo population compared to the total unsorted population based on log2 fold change of gene enrichment and p -value for enrichment generated by using the MAGeCK algorithm. (C) Bar chart shows the log2 fold change enrichment of targeting sgRNAs in CD2lo against the unsorted control population, which were reproduced in more than 1 of the 5 screen timepoints tested; each dot represents a separate timepoint. Data represent mean ± SD. (D) Flow cytometry histograms of surface CD2 expression, representative of two independent experiments, upon using two different sgRNA targeting BAP1 for knock out (BAP1 KO1 and KO2) and one sgRNA targeting CD2 (CD2 KO) compared to WT control and the corresponding geometric mean fluorescence intensity of each total population (gMFI). (E) A Western blot and corresponding bar charts show the total amount of BAP1 and CD2 protein normalized to beta-actin (b-actin) expression in experiments as in (D) using two different sgRNAs to knock out BAP1. (F) Histograms from two independent experiments are presented, here, show the surface CD2 expression upon three different sgRNA targeting SUZ12 for knock out (SUZ1 KO1, KO2, and KO3) compared to CD2 KO and WT control with corresponding gMFI of each total population. (G) A Western blot and corresponding bar charts show the total amount of SUZ12 protein normalized to GAPDH protein expression from experiments shown in (F). (H) Bar charts show the relative CD2 mRNA expression upon BAP1, SUZ12, and CD2 KO from experiments in D and F, compared to WT controls as quantified by real-time quantitative PCR; results from two different sgRNAs targeting each gene are shown. Also see and .

Article Snippet: CD2 (3A10B2) Mouse , Proteintech , Cat# 60005-1-Ig.

Techniques: CRISPR, Expressing, Generated, Control, Flow Cytometry, Knock-Out, Fluorescence, Western Blot, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: CD2 costimulation strength: A key regulator of T cell function and anti-tumor immunity that is epigenetically regulated

doi: 10.1016/j.isci.2025.113977

Figure Lengend Snippet: CD2 expression is coregulated with the expression of markers associated with T cell development, stemness, exhaustion, and metabolism (A) Volcano plot shows p values and fold changes in the expression of genes detected in BAP1 KO Jurkats relative to WT Jurkats. (B and C) Heatmaps of the log2 fold change (FC) expression of selected differentially expressed genes (DEGs) and their corresponding gene names identified in BAP1 KO Jurkats relative to WT control; these are categorized in T cell surface signaling molecules, adhesion molecules, transcription factors (TFs), and solute transporters in (B) and in factors associated with stemness and exhaustion of T cells in (C) categorized in two groups A and B as explained in section. (D and E) Bubble plots shows a selection of enriched pathways determined by performing gene ontology analysis (biological process pathways) in (D) and KEGG pathway analysis in (E) of DEGs in BAP1 KO T cells with a padjust<0.05. The gene count per pathway is represented by the size of the dot and the p value by the color of the dot. (F) A bar chart shows a selection of enriched pathways and the corresponding normalized enrichment score (NES) determined by performing gene set enrichment analysis (GSEA) using the C5 gene sets of the Molecular Signatures Database (MSigDB) and the same DEGs as in (D-E). (G) A selection of enriched gene signatures in BAP1 KO was identified upon GSEA using the C7 immunological signature gene sets in MSigDB. (H) GSEA in BAP1 KO T cells for a progenitor exhausted T cell gene signature generated from. Also see and .

Article Snippet: CD2 (3A10B2) Mouse , Proteintech , Cat# 60005-1-Ig.

Techniques: Expressing, Control, Selection, Generated

Journal: iScience

Article Title: CD2 costimulation strength: A key regulator of T cell function and anti-tumor immunity that is epigenetically regulated

doi: 10.1016/j.isci.2025.113977

Figure Lengend Snippet: BAP1 expression levels tune the expression of surface receptors in T cells (A) Flow cytometry contour plots show CD2 − CD3e expression profile during sgRNA BAP1 KO2 in Jurkat T cells at different timepoints of the culture and the gating to define different populations from one of the three representative experiments performed. Double-positive, DP: CD3 + CD2 + , single positive, SP: CD3 − CD2+/high, double-negative DN: CD3 − CD2-/low T cell subsets. (B) A Western blot and corresponding bar charts show the total amount of BAP1 protein normalized to beta-actin (b-actin) expression in experiments as in (A) at endpoint when culture showed no further deviation in ratio of DP, SP, and DN subsets. This is representative of two independent experiments. (C) Flow cytometry histograms (left) of CD2, CD28, CD11a, CD49a, and CD58 in DN, SP, DP BAP1KO2 T cell subsets and WT T cells and the corresponding percentage positive T cells for each marker is shown in bar chart (right) . This is representative of two independent experiments. (D) Right and left bar charts present the percentage of WT and mutant DNA sequences and the percentage of WT and mutant protein sequences with in-frame mutations (MT-IF) and in frameshift-missense mutations as part of the total DNA and protein sequences, respectively. This is shown for the three Jurkat FACSorted populations defined based on their CD2 − CD3e expression profile as described above, DN, SP, and DP. (E) Images of the modeled protein structures of WT BAP1 and mutant A BAP1 (one of the top three mutant protein sequences between the three Jurkat clones DN, SP, and DP; see also C. At the top, it is the model structure of the whole protein sequence, and underneath a zoom-in image of the region of the exon 8 that was targeted by the CRISP-CAS9-sgRNA complex. Also see .

Article Snippet: CD2 (3A10B2) Mouse , Proteintech , Cat# 60005-1-Ig.

Techniques: Expressing, Flow Cytometry, Western Blot, Marker, Mutagenesis, Clone Assay, Sequencing

Journal: iScience

Article Title: CD2 costimulation strength: A key regulator of T cell function and anti-tumor immunity that is epigenetically regulated

doi: 10.1016/j.isci.2025.113977

Figure Lengend Snippet: BAP1 is required for full upregulation and sustained expression of CD2, TCR, and PD-1, but not CD28 or CD226, following T cell activation (A) Flow cytometry histograms of CD2, CD28, CD226 and TCR expression in WT and DN BAP1KO Jurkat T cells in resting conditions and two days post-chemical cell activation by-passing the need to engage the TCR-CD3 complex, with low (0.6 ng/mL) and high (20 ng/mL) concentrations of phorbol myristate acetate (PMA), with low (0.1 μg/mL) and high ((1 μg/mL)) concentrations of ionomycin (ION) or a combination of high concentration of PMA with low or high concentration of ION. (B) Same as in (a) but 11–12 days post-chemical activation that includes a resting period of cells cultured in no stimulation conditions. A and B are representative of two independent experiments. The dot lines are aligned on the resting or lowPMA conditions of WT as a visual aid comparison reference. Also see and .

Article Snippet: CD2 (3A10B2) Mouse , Proteintech , Cat# 60005-1-Ig.

Techniques: Expressing, Activation Assay, Flow Cytometry, Concentration Assay, Cell Culture, Comparison

Journal: iScience

Article Title: CD2 costimulation strength: A key regulator of T cell function and anti-tumor immunity that is epigenetically regulated

doi: 10.1016/j.isci.2025.113977

Figure Lengend Snippet: Pharmacological inhibition of histone deacetylases partially rescues BAP1 loss-mediated CD2 repression (A) Flow cytometry histograms show the surface CD2 expression in DN BAP1KO T cells in control condition and upon treatment with three different PRC2 inhibitors in a dose-dependent manner, seven days post-initiation of treatment (PRC2i-1, GSK126; PRC2i-2, A-395; PRC2i-3, Tazemetostat). Results are from one/or two independent experiments performed for each inhibitor. (B) Flow cytometry histograms show the surface CD2 expression in DN BAP1KO T cells in control conditions and upon treatment with HDAC inhibitor, LMK235, in a dose-dependent manner, three and seven days post-initiation of treatment; a representative experiment is shown from two independent ones. WT represents CD2 expression in WT T cells in control, untreated conditions. Also see .

Article Snippet: CD2 (3A10B2) Mouse , Proteintech , Cat# 60005-1-Ig.

Techniques: Inhibition, Flow Cytometry, Expressing, Control

Journal: iScience

Article Title: CD2 costimulation strength: A key regulator of T cell function and anti-tumor immunity that is epigenetically regulated

doi: 10.1016/j.isci.2025.113977

Figure Lengend Snippet: Magnitude of T cell proliferation and IFN-γ production correlates with CD2 costimulation strength Representative flow cytometry histograms (left) from one healthy volunteer, illustrating cell divisions of PB CFSE-labelled naive (A) and memory (B) CD4 + T cells, gated on divided cells and corresponding plots (right) of the percentage decrease in proliferation index and replication index at titrated levels of CD2 costimulation strength (i-vi), 5-day post in-vitro activation from 7 donors (each donor is shown with a different color). From experiments as in (Α and B), representative histograms (left) of CD25 expression in naive (C) and memory (D) CD4 + T cells 2 days post in-vitro activation, and corresponding plots (right) show CD25 expression against PI or RI expression for 8 and 3 donors, respectively, (each donor is shown with a different color and all parameters normalized to value in highest CD2 costimulation strength condition - black datapoint - (i) to allow the presentation of all healthy donors samples in one plot). (E) Histograms of intracellular levels of IFN-γ (top) and IL-2 (bottom) expression of naive CD4 + T cells differentiated to Th1 cells in-vitro, 7 days post-activation, and (F) Quantification of percentages of 7 populations of cytokine producing subsets, as gated in representative IL-2 vs. IFN-γ dot plots, in the presence of titrated levels of CD2 costimulation strength (i-vi); a representative donor shown from two independent healthy donor experiments performed is shown in (E) and (F). Also see .

Article Snippet: CD2 (3A10B2) Mouse , Proteintech , Cat# 60005-1-Ig.

Techniques: Flow Cytometry, In Vitro, Activation Assay, Expressing