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anti mouse pdpn gp38  (Bio X Cell)

 
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    Bio X Cell anti mouse pdpn gp38
    ( A ) Gating strategy for FACS of CD11b + F4/80 + and CD11b + F4/80 – cells from the peritoneal cells of WT and Adap –/– mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). RNA-Seq analysis identified DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. ( B ) Schematic of RNA-Seq analysis of WT and Adap –/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap –/– PMs from E. coli –infected mice (209 DEGs) and WT versus Adap –/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). ( C ) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B . ( D – F ) <t>PDPN</t> expression in WT and Adap –/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting ( D ), flow cytometry ( E ), and qPCR ( F , n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( G ) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAP KD ) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. ( H ) Pdpn mRNA expression in WT and Adap –/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR ( n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( I ) ADAP KD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.
    Anti Mouse Pdpn Gp38, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse pdpn gp38/product/Bio X Cell
    Average 94 stars, based on 18 article reviews
    anti mouse pdpn gp38 - by Bioz Stars, 2026-02
    94/100 stars

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    1) Product Images from "Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis"

    Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

    Journal: JCI Insight

    doi: 10.1172/jci.insight.186456

    ( A ) Gating strategy for FACS of CD11b + F4/80 + and CD11b + F4/80 – cells from the peritoneal cells of WT and Adap –/– mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). RNA-Seq analysis identified DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. ( B ) Schematic of RNA-Seq analysis of WT and Adap –/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap –/– PMs from E. coli –infected mice (209 DEGs) and WT versus Adap –/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). ( C ) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B . ( D – F ) PDPN expression in WT and Adap –/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting ( D ), flow cytometry ( E ), and qPCR ( F , n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( G ) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAP KD ) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. ( H ) Pdpn mRNA expression in WT and Adap –/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR ( n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( I ) ADAP KD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.
    Figure Legend Snippet: ( A ) Gating strategy for FACS of CD11b + F4/80 + and CD11b + F4/80 – cells from the peritoneal cells of WT and Adap –/– mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). RNA-Seq analysis identified DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. ( B ) Schematic of RNA-Seq analysis of WT and Adap –/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap –/– PMs from E. coli –infected mice (209 DEGs) and WT versus Adap –/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). ( C ) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B . ( D – F ) PDPN expression in WT and Adap –/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting ( D ), flow cytometry ( E ), and qPCR ( F , n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( G ) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAP KD ) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. ( H ) Pdpn mRNA expression in WT and Adap –/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR ( n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( I ) ADAP KD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.

    Techniques Used: Injection, RNA Sequencing, In Vitro, Infection, Expressing, Western Blot, Flow Cytometry, Comparison, Knockdown, Transduction, Over Expression, Plasmid Preparation, Control, Derivative Assay

    ( A ) Vertical scatter plots summarizing the mRNA expression levels of Adap , Pdpn , and Il6 as measured by qPCR analysis of peritoneal cells from WT mice 18 hours after injection of saline or E . coli (2 × 10 7 CFU, i.p.) ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( B ) Vertical scatter plots showing the expression of Adap and Pdpn genes (expressed as RNA-Seq reads) extracted from RNA-Seq data of peritoneal cells from LPS-induced peritonitis in mice ( n = 6 each, unpaired t test). ( C ) The expression of Adap and Pdpn in the peritoneal cells of E . coli –infected mice was analyzed using a published database (GEO accession no. GSE34114, naive 2 hours, n = 2; naive 18 hours, n = 3; E . coli 1 hour, n = 2; E . coli 2 hours, n = 2; E . coli 18 hours, n = 3). Gene expression was analyzed in GEO2R. ( D and E ) Peritoneal exudate cells were isolated from WT, Adap –/– , and Skap1 –/– mice 18 hours after injection of E . coli (2 × 10 7 CFU, i.p.), and CD11b + F4/80 + PDPN hi macrophages were analyzed by flow cytometry. Left panels: Representative contour plots showing the frequency of PDPN hi macrophages in the peritoneal cavity of WT, Adap –/– , and Skap1 –/– mice at 18 hours after E . coli injection. Right panels: Bar graphs showing the percentage of PDPN hi macrophages ( D , n = 6 each; E , n = 3 each; unpaired t test).
    Figure Legend Snippet: ( A ) Vertical scatter plots summarizing the mRNA expression levels of Adap , Pdpn , and Il6 as measured by qPCR analysis of peritoneal cells from WT mice 18 hours after injection of saline or E . coli (2 × 10 7 CFU, i.p.) ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( B ) Vertical scatter plots showing the expression of Adap and Pdpn genes (expressed as RNA-Seq reads) extracted from RNA-Seq data of peritoneal cells from LPS-induced peritonitis in mice ( n = 6 each, unpaired t test). ( C ) The expression of Adap and Pdpn in the peritoneal cells of E . coli –infected mice was analyzed using a published database (GEO accession no. GSE34114, naive 2 hours, n = 2; naive 18 hours, n = 3; E . coli 1 hour, n = 2; E . coli 2 hours, n = 2; E . coli 18 hours, n = 3). Gene expression was analyzed in GEO2R. ( D and E ) Peritoneal exudate cells were isolated from WT, Adap –/– , and Skap1 –/– mice 18 hours after injection of E . coli (2 × 10 7 CFU, i.p.), and CD11b + F4/80 + PDPN hi macrophages were analyzed by flow cytometry. Left panels: Representative contour plots showing the frequency of PDPN hi macrophages in the peritoneal cavity of WT, Adap –/– , and Skap1 –/– mice at 18 hours after E . coli injection. Right panels: Bar graphs showing the percentage of PDPN hi macrophages ( D , n = 6 each; E , n = 3 each; unpaired t test).

    Techniques Used: Expressing, Injection, Saline, RNA Sequencing, Infection, Gene Expression, Isolation, Flow Cytometry

    ( A ) RNA-Seq analysis of CD11b + F4/80 + PDPN hi and CD11b + F4/80 + PDPN lo PMs sorted from WT mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.), showing the numbers of upregulated and downregulated DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR). ( B ) Top 10 KEGG pathways identified from DEGs in A by KEGG enrichment analysis. ( C ) Heatmaps showing the DEGs of the chemokine-related genes in CD11b + F4/80 + PDPN hi PMs, compared with CD11b + F4/80 + PDPN lo , grouped by hierarchical clustering analysis of the RNA-Seq data. ( D and E ) qPCR analysis of M2-related chemokine DEGs ( D ) and polarization markers ( E ) in PDPN hi versus PDPN lo PMs from WT septic mice ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( F ) Flow cytometric analysis of E . coli –GFP uptake by CD11b + F4/80 + PDPN lo and CD11b + F4/80 + PDPN hi PMs in LPS-treated septic WT mice. GFP fluorescence (mean fluorescence intensity) was compared ( n = 3 each, unpaired t test). ( G ) GSEA histogram for the “phagosome” gene set in PDPN hi versus PDPN lo PMs. The normalized enrichment score (NES) and FDR q value are indicated. ( H ) The mRNA levels of Cd36 and Marco in PDPN hi and PDPN lo PMs from WT septic mice were determined using qPCR ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( I ) Kaplan-Meier survival analysis of WT and Adap –/– mice injected with anti-PDPN blocking antibodies (100 μg/mouse, i.v.) 2 hours after E . coli infection (2 × 10 7 CFU, i.p.) (WT E . coli , n = 15; Adap –/– E . coli , n = 10; WT E . coli –anti-PDPN, n = 16; Adap –/– E . coli –anti-PDPN, n = 11). Log-rank test was used to compare survival curve.
    Figure Legend Snippet: ( A ) RNA-Seq analysis of CD11b + F4/80 + PDPN hi and CD11b + F4/80 + PDPN lo PMs sorted from WT mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.), showing the numbers of upregulated and downregulated DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR). ( B ) Top 10 KEGG pathways identified from DEGs in A by KEGG enrichment analysis. ( C ) Heatmaps showing the DEGs of the chemokine-related genes in CD11b + F4/80 + PDPN hi PMs, compared with CD11b + F4/80 + PDPN lo , grouped by hierarchical clustering analysis of the RNA-Seq data. ( D and E ) qPCR analysis of M2-related chemokine DEGs ( D ) and polarization markers ( E ) in PDPN hi versus PDPN lo PMs from WT septic mice ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( F ) Flow cytometric analysis of E . coli –GFP uptake by CD11b + F4/80 + PDPN lo and CD11b + F4/80 + PDPN hi PMs in LPS-treated septic WT mice. GFP fluorescence (mean fluorescence intensity) was compared ( n = 3 each, unpaired t test). ( G ) GSEA histogram for the “phagosome” gene set in PDPN hi versus PDPN lo PMs. The normalized enrichment score (NES) and FDR q value are indicated. ( H ) The mRNA levels of Cd36 and Marco in PDPN hi and PDPN lo PMs from WT septic mice were determined using qPCR ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( I ) Kaplan-Meier survival analysis of WT and Adap –/– mice injected with anti-PDPN blocking antibodies (100 μg/mouse, i.v.) 2 hours after E . coli infection (2 × 10 7 CFU, i.p.) (WT E . coli , n = 15; Adap –/– E . coli , n = 10; WT E . coli –anti-PDPN, n = 16; Adap –/– E . coli –anti-PDPN, n = 11). Log-rank test was used to compare survival curve.

    Techniques Used: RNA Sequencing, Injection, Fluorescence, Blocking Assay, Infection

    ( A – D ) WT PMs were pretreated with resatorvid (0.1, 0.5, and 1 μM), IKK-16 (0.1, 0.3, and 0.5 μM), Bay 11-7085 (0.5, 1, and 5 μM), SB203580 (1, 5, and 10 μM), or SP600125 (1, 10, and 20 μM) for 1 hour, followed by LPS stimulation (100 ng/mL) for 24 hours ( A , C , and D ) or 12 hours ( B ). ADAP and PDPN expression was analyzed by Western blotting and qPCR ( B , n = 3 each, 1-way ANOVA, Tukey’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels ( D ). ( E ) RAW264.7 cells transduced with ADAP were treated with LPS (100 ng/mL, 24 hours) in the presence of IKK-16 (0.5 μM). ADAP and PDPN expression was analyzed by Western blotting. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels. ( F ) Schematic of kinase inhibitors screening and summary of inhibitors blocking LPS-induced PDPN upregulation. ( G ) WT PMs pretreated with ibrutinib (1, 5, and 10 μM) or dasatinib (0.01, 0.1, and 1 μM) for 1 hour were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was assessed by Western blotting. ( H ) MS confirmed LPS-induced (100 ng/mL, 1 hour) ADAP phosphorylation at Y 571 in PMs. MS/MS spectra of the phosphorylated peptide (TTAVEIDYDSLKR) are shown. ( I ) ADAP KD RAW264.7 cells reconstituted with empty vector (EV), ADAP-WT, or ADAP (Y571F) were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was analyzed by Western blotting. ( J ) CD11b + F4/80 + PDPN hi (C2) and CD11b + F4/80 + PDPN lo (C1) PMs were sorted from WT septic mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). Lysates were immunoprecipitated with anti-ADAP and immunoblotted for p-Tyr 100 and ADAP.
    Figure Legend Snippet: ( A – D ) WT PMs were pretreated with resatorvid (0.1, 0.5, and 1 μM), IKK-16 (0.1, 0.3, and 0.5 μM), Bay 11-7085 (0.5, 1, and 5 μM), SB203580 (1, 5, and 10 μM), or SP600125 (1, 10, and 20 μM) for 1 hour, followed by LPS stimulation (100 ng/mL) for 24 hours ( A , C , and D ) or 12 hours ( B ). ADAP and PDPN expression was analyzed by Western blotting and qPCR ( B , n = 3 each, 1-way ANOVA, Tukey’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels ( D ). ( E ) RAW264.7 cells transduced with ADAP were treated with LPS (100 ng/mL, 24 hours) in the presence of IKK-16 (0.5 μM). ADAP and PDPN expression was analyzed by Western blotting. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels. ( F ) Schematic of kinase inhibitors screening and summary of inhibitors blocking LPS-induced PDPN upregulation. ( G ) WT PMs pretreated with ibrutinib (1, 5, and 10 μM) or dasatinib (0.01, 0.1, and 1 μM) for 1 hour were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was assessed by Western blotting. ( H ) MS confirmed LPS-induced (100 ng/mL, 1 hour) ADAP phosphorylation at Y 571 in PMs. MS/MS spectra of the phosphorylated peptide (TTAVEIDYDSLKR) are shown. ( I ) ADAP KD RAW264.7 cells reconstituted with empty vector (EV), ADAP-WT, or ADAP (Y571F) were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was analyzed by Western blotting. ( J ) CD11b + F4/80 + PDPN hi (C2) and CD11b + F4/80 + PDPN lo (C1) PMs were sorted from WT septic mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). Lysates were immunoprecipitated with anti-ADAP and immunoblotted for p-Tyr 100 and ADAP.

    Techniques Used: Expressing, Western Blot, Comparison, Derivative Assay, Transduction, Blocking Assay, Tandem Mass Spectroscopy, Plasmid Preparation, Injection, Immunoprecipitation

    ( A ) PDPN expression in PMs pretreated with BP-1-102 (0.5, 1, and 5 μM) or fludarabine (10, 50, and 100 μM) for 1 hour and stimulated with LPS (100 ng/mL, 24 hours), analyzed by Western blotting and densitometry normalized to α-tubulin ( n = 6 each, 1-way ANOVA, Tukey’s multiple-comparison test). ( B ) STAT3 binding motifs in the Pdpn promoter and CUT & RUN qPCR showing STAT3 enrichment at site 2 in untreated or LPS-treated PMs (100 ng/mL, 1 hour) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( C ) Luciferase activity of Pdpn -WT-Luc or Pdpn -Mut-Luc in WT or ADAP KD RAW264.7 cells following LPS stimulation (100 ng/mL, 6 hours) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( D ) EMSA showing STAT3-DNA binding in nuclear extracts of WT or ADAP KD RAW264.7 cells after LPS stimulation (100 ng/mL, 1 hour). ( E ) Flow cytometry of CD11b + F4/80 + PDPN hi PMs in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (2 × 10 7 CFU, i.p.) ( n = 5 each, unpaired t test). ( F and G ) Western blot of PDPN, p-STAT3, and STAT3 in LPS-treated PMs (100 ng/mL) with or without mTOR inhibitors or rapamycin (1 μM). ( H ) LPS-stimulated PMs (100 ng/mL, 1 hour) pretreated with rapamycin (1 μM, 1 hour) were immunoprecipitated with anti-ADAP and immunoblotted for STAT3. ( I ) PDPN expression in WT and Adap –/– PMs treated with LPS (10 ng/mL) and MHY1485 (10 μM, 24 hours) was analyzed by Western blotting. ( J and K ) Kaplan-Meier survival curves and bacterial burden in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (5 × 10 7 CFU, i.p.), showing survival ( n = 15 each, log-rank test) and bacterial load in peritoneal lavage fluid and blood at 18 hours ( n = 5 each; unpaired t test).
    Figure Legend Snippet: ( A ) PDPN expression in PMs pretreated with BP-1-102 (0.5, 1, and 5 μM) or fludarabine (10, 50, and 100 μM) for 1 hour and stimulated with LPS (100 ng/mL, 24 hours), analyzed by Western blotting and densitometry normalized to α-tubulin ( n = 6 each, 1-way ANOVA, Tukey’s multiple-comparison test). ( B ) STAT3 binding motifs in the Pdpn promoter and CUT & RUN qPCR showing STAT3 enrichment at site 2 in untreated or LPS-treated PMs (100 ng/mL, 1 hour) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( C ) Luciferase activity of Pdpn -WT-Luc or Pdpn -Mut-Luc in WT or ADAP KD RAW264.7 cells following LPS stimulation (100 ng/mL, 6 hours) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( D ) EMSA showing STAT3-DNA binding in nuclear extracts of WT or ADAP KD RAW264.7 cells after LPS stimulation (100 ng/mL, 1 hour). ( E ) Flow cytometry of CD11b + F4/80 + PDPN hi PMs in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (2 × 10 7 CFU, i.p.) ( n = 5 each, unpaired t test). ( F and G ) Western blot of PDPN, p-STAT3, and STAT3 in LPS-treated PMs (100 ng/mL) with or without mTOR inhibitors or rapamycin (1 μM). ( H ) LPS-stimulated PMs (100 ng/mL, 1 hour) pretreated with rapamycin (1 μM, 1 hour) were immunoprecipitated with anti-ADAP and immunoblotted for STAT3. ( I ) PDPN expression in WT and Adap –/– PMs treated with LPS (10 ng/mL) and MHY1485 (10 μM, 24 hours) was analyzed by Western blotting. ( J and K ) Kaplan-Meier survival curves and bacterial burden in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (5 × 10 7 CFU, i.p.), showing survival ( n = 15 each, log-rank test) and bacterial load in peritoneal lavage fluid and blood at 18 hours ( n = 5 each; unpaired t test).

    Techniques Used: Expressing, Western Blot, Comparison, Binding Assay, Luciferase, Activity Assay, Flow Cytometry, Infection, Immunoprecipitation

    ADAP is upregulated in sepsis and serves as an LPS stimulus–responsive protein that governs the upregulation of PDPN to form a distinct PDPN hi PM subset during sepsis, which displays a phenotype closely akin to M2 macrophages with enhanced phagocytic activity and provides enhanced protection against sepsis in the host. Mechanistically, while TLR4 signaling stimulates the upregulation of ADAP via the NF-κB pathway, TLR4-triggered phosphorylation of ADAP at Y 571 by BTK primes the subsequent direct phosphorylation of STAT3 by mTOR, which transactivates PDPN transcription.
    Figure Legend Snippet: ADAP is upregulated in sepsis and serves as an LPS stimulus–responsive protein that governs the upregulation of PDPN to form a distinct PDPN hi PM subset during sepsis, which displays a phenotype closely akin to M2 macrophages with enhanced phagocytic activity and provides enhanced protection against sepsis in the host. Mechanistically, while TLR4 signaling stimulates the upregulation of ADAP via the NF-κB pathway, TLR4-triggered phosphorylation of ADAP at Y 571 by BTK primes the subsequent direct phosphorylation of STAT3 by mTOR, which transactivates PDPN transcription.

    Techniques Used: Activity Assay



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    anti mouse pdpn gp38  (Bio X Cell)
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    ( A ) Gating strategy for FACS of CD11b + F4/80 + and CD11b + F4/80 – cells from the peritoneal cells of WT and Adap –/– mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). RNA-Seq analysis identified DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. ( B ) Schematic of RNA-Seq analysis of WT and Adap –/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap –/– PMs from E. coli –infected mice (209 DEGs) and WT versus Adap –/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). ( C ) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B . ( D – F ) <t>PDPN</t> expression in WT and Adap –/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting ( D ), flow cytometry ( E ), and qPCR ( F , n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( G ) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAP KD ) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. ( H ) Pdpn mRNA expression in WT and Adap –/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR ( n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( I ) ADAP KD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.
    Anti Mouse Pdpn Gp38, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse pdpn gp38/product/Bio X Cell
    Average 94 stars, based on 1 article reviews
    anti mouse pdpn gp38 - by Bioz Stars, 2026-02
    94/100 stars
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    ( A ) Gating strategy for FACS of CD11b + F4/80 + and CD11b + F4/80 – cells from the peritoneal cells of WT and Adap –/– mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). RNA-Seq analysis identified DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. ( B ) Schematic of RNA-Seq analysis of WT and Adap –/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap –/– PMs from E. coli –infected mice (209 DEGs) and WT versus Adap –/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). ( C ) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B . ( D – F ) PDPN expression in WT and Adap –/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting ( D ), flow cytometry ( E ), and qPCR ( F , n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( G ) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAP KD ) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. ( H ) Pdpn mRNA expression in WT and Adap –/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR ( n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( I ) ADAP KD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.

    Journal: JCI Insight

    Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

    doi: 10.1172/jci.insight.186456

    Figure Lengend Snippet: ( A ) Gating strategy for FACS of CD11b + F4/80 + and CD11b + F4/80 – cells from the peritoneal cells of WT and Adap –/– mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). RNA-Seq analysis identified DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. ( B ) Schematic of RNA-Seq analysis of WT and Adap –/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap –/– PMs from E. coli –infected mice (209 DEGs) and WT versus Adap –/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). ( C ) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B . ( D – F ) PDPN expression in WT and Adap –/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting ( D ), flow cytometry ( E ), and qPCR ( F , n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( G ) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAP KD ) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. ( H ) Pdpn mRNA expression in WT and Adap –/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR ( n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( I ) ADAP KD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.

    Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

    Techniques: Injection, RNA Sequencing, In Vitro, Infection, Expressing, Western Blot, Flow Cytometry, Comparison, Knockdown, Transduction, Over Expression, Plasmid Preparation, Control, Derivative Assay

    ( A ) Vertical scatter plots summarizing the mRNA expression levels of Adap , Pdpn , and Il6 as measured by qPCR analysis of peritoneal cells from WT mice 18 hours after injection of saline or E . coli (2 × 10 7 CFU, i.p.) ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( B ) Vertical scatter plots showing the expression of Adap and Pdpn genes (expressed as RNA-Seq reads) extracted from RNA-Seq data of peritoneal cells from LPS-induced peritonitis in mice ( n = 6 each, unpaired t test). ( C ) The expression of Adap and Pdpn in the peritoneal cells of E . coli –infected mice was analyzed using a published database (GEO accession no. GSE34114, naive 2 hours, n = 2; naive 18 hours, n = 3; E . coli 1 hour, n = 2; E . coli 2 hours, n = 2; E . coli 18 hours, n = 3). Gene expression was analyzed in GEO2R. ( D and E ) Peritoneal exudate cells were isolated from WT, Adap –/– , and Skap1 –/– mice 18 hours after injection of E . coli (2 × 10 7 CFU, i.p.), and CD11b + F4/80 + PDPN hi macrophages were analyzed by flow cytometry. Left panels: Representative contour plots showing the frequency of PDPN hi macrophages in the peritoneal cavity of WT, Adap –/– , and Skap1 –/– mice at 18 hours after E . coli injection. Right panels: Bar graphs showing the percentage of PDPN hi macrophages ( D , n = 6 each; E , n = 3 each; unpaired t test).

    Journal: JCI Insight

    Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

    doi: 10.1172/jci.insight.186456

    Figure Lengend Snippet: ( A ) Vertical scatter plots summarizing the mRNA expression levels of Adap , Pdpn , and Il6 as measured by qPCR analysis of peritoneal cells from WT mice 18 hours after injection of saline or E . coli (2 × 10 7 CFU, i.p.) ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( B ) Vertical scatter plots showing the expression of Adap and Pdpn genes (expressed as RNA-Seq reads) extracted from RNA-Seq data of peritoneal cells from LPS-induced peritonitis in mice ( n = 6 each, unpaired t test). ( C ) The expression of Adap and Pdpn in the peritoneal cells of E . coli –infected mice was analyzed using a published database (GEO accession no. GSE34114, naive 2 hours, n = 2; naive 18 hours, n = 3; E . coli 1 hour, n = 2; E . coli 2 hours, n = 2; E . coli 18 hours, n = 3). Gene expression was analyzed in GEO2R. ( D and E ) Peritoneal exudate cells were isolated from WT, Adap –/– , and Skap1 –/– mice 18 hours after injection of E . coli (2 × 10 7 CFU, i.p.), and CD11b + F4/80 + PDPN hi macrophages were analyzed by flow cytometry. Left panels: Representative contour plots showing the frequency of PDPN hi macrophages in the peritoneal cavity of WT, Adap –/– , and Skap1 –/– mice at 18 hours after E . coli injection. Right panels: Bar graphs showing the percentage of PDPN hi macrophages ( D , n = 6 each; E , n = 3 each; unpaired t test).

    Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

    Techniques: Expressing, Injection, Saline, RNA Sequencing, Infection, Gene Expression, Isolation, Flow Cytometry

    ( A ) RNA-Seq analysis of CD11b + F4/80 + PDPN hi and CD11b + F4/80 + PDPN lo PMs sorted from WT mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.), showing the numbers of upregulated and downregulated DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR). ( B ) Top 10 KEGG pathways identified from DEGs in A by KEGG enrichment analysis. ( C ) Heatmaps showing the DEGs of the chemokine-related genes in CD11b + F4/80 + PDPN hi PMs, compared with CD11b + F4/80 + PDPN lo , grouped by hierarchical clustering analysis of the RNA-Seq data. ( D and E ) qPCR analysis of M2-related chemokine DEGs ( D ) and polarization markers ( E ) in PDPN hi versus PDPN lo PMs from WT septic mice ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( F ) Flow cytometric analysis of E . coli –GFP uptake by CD11b + F4/80 + PDPN lo and CD11b + F4/80 + PDPN hi PMs in LPS-treated septic WT mice. GFP fluorescence (mean fluorescence intensity) was compared ( n = 3 each, unpaired t test). ( G ) GSEA histogram for the “phagosome” gene set in PDPN hi versus PDPN lo PMs. The normalized enrichment score (NES) and FDR q value are indicated. ( H ) The mRNA levels of Cd36 and Marco in PDPN hi and PDPN lo PMs from WT septic mice were determined using qPCR ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( I ) Kaplan-Meier survival analysis of WT and Adap –/– mice injected with anti-PDPN blocking antibodies (100 μg/mouse, i.v.) 2 hours after E . coli infection (2 × 10 7 CFU, i.p.) (WT E . coli , n = 15; Adap –/– E . coli , n = 10; WT E . coli –anti-PDPN, n = 16; Adap –/– E . coli –anti-PDPN, n = 11). Log-rank test was used to compare survival curve.

    Journal: JCI Insight

    Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

    doi: 10.1172/jci.insight.186456

    Figure Lengend Snippet: ( A ) RNA-Seq analysis of CD11b + F4/80 + PDPN hi and CD11b + F4/80 + PDPN lo PMs sorted from WT mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.), showing the numbers of upregulated and downregulated DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR). ( B ) Top 10 KEGG pathways identified from DEGs in A by KEGG enrichment analysis. ( C ) Heatmaps showing the DEGs of the chemokine-related genes in CD11b + F4/80 + PDPN hi PMs, compared with CD11b + F4/80 + PDPN lo , grouped by hierarchical clustering analysis of the RNA-Seq data. ( D and E ) qPCR analysis of M2-related chemokine DEGs ( D ) and polarization markers ( E ) in PDPN hi versus PDPN lo PMs from WT septic mice ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( F ) Flow cytometric analysis of E . coli –GFP uptake by CD11b + F4/80 + PDPN lo and CD11b + F4/80 + PDPN hi PMs in LPS-treated septic WT mice. GFP fluorescence (mean fluorescence intensity) was compared ( n = 3 each, unpaired t test). ( G ) GSEA histogram for the “phagosome” gene set in PDPN hi versus PDPN lo PMs. The normalized enrichment score (NES) and FDR q value are indicated. ( H ) The mRNA levels of Cd36 and Marco in PDPN hi and PDPN lo PMs from WT septic mice were determined using qPCR ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( I ) Kaplan-Meier survival analysis of WT and Adap –/– mice injected with anti-PDPN blocking antibodies (100 μg/mouse, i.v.) 2 hours after E . coli infection (2 × 10 7 CFU, i.p.) (WT E . coli , n = 15; Adap –/– E . coli , n = 10; WT E . coli –anti-PDPN, n = 16; Adap –/– E . coli –anti-PDPN, n = 11). Log-rank test was used to compare survival curve.

    Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

    Techniques: RNA Sequencing, Injection, Fluorescence, Blocking Assay, Infection

    ( A – D ) WT PMs were pretreated with resatorvid (0.1, 0.5, and 1 μM), IKK-16 (0.1, 0.3, and 0.5 μM), Bay 11-7085 (0.5, 1, and 5 μM), SB203580 (1, 5, and 10 μM), or SP600125 (1, 10, and 20 μM) for 1 hour, followed by LPS stimulation (100 ng/mL) for 24 hours ( A , C , and D ) or 12 hours ( B ). ADAP and PDPN expression was analyzed by Western blotting and qPCR ( B , n = 3 each, 1-way ANOVA, Tukey’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels ( D ). ( E ) RAW264.7 cells transduced with ADAP were treated with LPS (100 ng/mL, 24 hours) in the presence of IKK-16 (0.5 μM). ADAP and PDPN expression was analyzed by Western blotting. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels. ( F ) Schematic of kinase inhibitors screening and summary of inhibitors blocking LPS-induced PDPN upregulation. ( G ) WT PMs pretreated with ibrutinib (1, 5, and 10 μM) or dasatinib (0.01, 0.1, and 1 μM) for 1 hour were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was assessed by Western blotting. ( H ) MS confirmed LPS-induced (100 ng/mL, 1 hour) ADAP phosphorylation at Y 571 in PMs. MS/MS spectra of the phosphorylated peptide (TTAVEIDYDSLKR) are shown. ( I ) ADAP KD RAW264.7 cells reconstituted with empty vector (EV), ADAP-WT, or ADAP (Y571F) were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was analyzed by Western blotting. ( J ) CD11b + F4/80 + PDPN hi (C2) and CD11b + F4/80 + PDPN lo (C1) PMs were sorted from WT septic mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). Lysates were immunoprecipitated with anti-ADAP and immunoblotted for p-Tyr 100 and ADAP.

    Journal: JCI Insight

    Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

    doi: 10.1172/jci.insight.186456

    Figure Lengend Snippet: ( A – D ) WT PMs were pretreated with resatorvid (0.1, 0.5, and 1 μM), IKK-16 (0.1, 0.3, and 0.5 μM), Bay 11-7085 (0.5, 1, and 5 μM), SB203580 (1, 5, and 10 μM), or SP600125 (1, 10, and 20 μM) for 1 hour, followed by LPS stimulation (100 ng/mL) for 24 hours ( A , C , and D ) or 12 hours ( B ). ADAP and PDPN expression was analyzed by Western blotting and qPCR ( B , n = 3 each, 1-way ANOVA, Tukey’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels ( D ). ( E ) RAW264.7 cells transduced with ADAP were treated with LPS (100 ng/mL, 24 hours) in the presence of IKK-16 (0.5 μM). ADAP and PDPN expression was analyzed by Western blotting. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels. ( F ) Schematic of kinase inhibitors screening and summary of inhibitors blocking LPS-induced PDPN upregulation. ( G ) WT PMs pretreated with ibrutinib (1, 5, and 10 μM) or dasatinib (0.01, 0.1, and 1 μM) for 1 hour were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was assessed by Western blotting. ( H ) MS confirmed LPS-induced (100 ng/mL, 1 hour) ADAP phosphorylation at Y 571 in PMs. MS/MS spectra of the phosphorylated peptide (TTAVEIDYDSLKR) are shown. ( I ) ADAP KD RAW264.7 cells reconstituted with empty vector (EV), ADAP-WT, or ADAP (Y571F) were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was analyzed by Western blotting. ( J ) CD11b + F4/80 + PDPN hi (C2) and CD11b + F4/80 + PDPN lo (C1) PMs were sorted from WT septic mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). Lysates were immunoprecipitated with anti-ADAP and immunoblotted for p-Tyr 100 and ADAP.

    Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

    Techniques: Expressing, Western Blot, Comparison, Derivative Assay, Transduction, Blocking Assay, Tandem Mass Spectroscopy, Plasmid Preparation, Injection, Immunoprecipitation

    ( A ) PDPN expression in PMs pretreated with BP-1-102 (0.5, 1, and 5 μM) or fludarabine (10, 50, and 100 μM) for 1 hour and stimulated with LPS (100 ng/mL, 24 hours), analyzed by Western blotting and densitometry normalized to α-tubulin ( n = 6 each, 1-way ANOVA, Tukey’s multiple-comparison test). ( B ) STAT3 binding motifs in the Pdpn promoter and CUT & RUN qPCR showing STAT3 enrichment at site 2 in untreated or LPS-treated PMs (100 ng/mL, 1 hour) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( C ) Luciferase activity of Pdpn -WT-Luc or Pdpn -Mut-Luc in WT or ADAP KD RAW264.7 cells following LPS stimulation (100 ng/mL, 6 hours) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( D ) EMSA showing STAT3-DNA binding in nuclear extracts of WT or ADAP KD RAW264.7 cells after LPS stimulation (100 ng/mL, 1 hour). ( E ) Flow cytometry of CD11b + F4/80 + PDPN hi PMs in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (2 × 10 7 CFU, i.p.) ( n = 5 each, unpaired t test). ( F and G ) Western blot of PDPN, p-STAT3, and STAT3 in LPS-treated PMs (100 ng/mL) with or without mTOR inhibitors or rapamycin (1 μM). ( H ) LPS-stimulated PMs (100 ng/mL, 1 hour) pretreated with rapamycin (1 μM, 1 hour) were immunoprecipitated with anti-ADAP and immunoblotted for STAT3. ( I ) PDPN expression in WT and Adap –/– PMs treated with LPS (10 ng/mL) and MHY1485 (10 μM, 24 hours) was analyzed by Western blotting. ( J and K ) Kaplan-Meier survival curves and bacterial burden in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (5 × 10 7 CFU, i.p.), showing survival ( n = 15 each, log-rank test) and bacterial load in peritoneal lavage fluid and blood at 18 hours ( n = 5 each; unpaired t test).

    Journal: JCI Insight

    Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

    doi: 10.1172/jci.insight.186456

    Figure Lengend Snippet: ( A ) PDPN expression in PMs pretreated with BP-1-102 (0.5, 1, and 5 μM) or fludarabine (10, 50, and 100 μM) for 1 hour and stimulated with LPS (100 ng/mL, 24 hours), analyzed by Western blotting and densitometry normalized to α-tubulin ( n = 6 each, 1-way ANOVA, Tukey’s multiple-comparison test). ( B ) STAT3 binding motifs in the Pdpn promoter and CUT & RUN qPCR showing STAT3 enrichment at site 2 in untreated or LPS-treated PMs (100 ng/mL, 1 hour) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( C ) Luciferase activity of Pdpn -WT-Luc or Pdpn -Mut-Luc in WT or ADAP KD RAW264.7 cells following LPS stimulation (100 ng/mL, 6 hours) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( D ) EMSA showing STAT3-DNA binding in nuclear extracts of WT or ADAP KD RAW264.7 cells after LPS stimulation (100 ng/mL, 1 hour). ( E ) Flow cytometry of CD11b + F4/80 + PDPN hi PMs in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (2 × 10 7 CFU, i.p.) ( n = 5 each, unpaired t test). ( F and G ) Western blot of PDPN, p-STAT3, and STAT3 in LPS-treated PMs (100 ng/mL) with or without mTOR inhibitors or rapamycin (1 μM). ( H ) LPS-stimulated PMs (100 ng/mL, 1 hour) pretreated with rapamycin (1 μM, 1 hour) were immunoprecipitated with anti-ADAP and immunoblotted for STAT3. ( I ) PDPN expression in WT and Adap –/– PMs treated with LPS (10 ng/mL) and MHY1485 (10 μM, 24 hours) was analyzed by Western blotting. ( J and K ) Kaplan-Meier survival curves and bacterial burden in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (5 × 10 7 CFU, i.p.), showing survival ( n = 15 each, log-rank test) and bacterial load in peritoneal lavage fluid and blood at 18 hours ( n = 5 each; unpaired t test).

    Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

    Techniques: Expressing, Western Blot, Comparison, Binding Assay, Luciferase, Activity Assay, Flow Cytometry, Infection, Immunoprecipitation

    ADAP is upregulated in sepsis and serves as an LPS stimulus–responsive protein that governs the upregulation of PDPN to form a distinct PDPN hi PM subset during sepsis, which displays a phenotype closely akin to M2 macrophages with enhanced phagocytic activity and provides enhanced protection against sepsis in the host. Mechanistically, while TLR4 signaling stimulates the upregulation of ADAP via the NF-κB pathway, TLR4-triggered phosphorylation of ADAP at Y 571 by BTK primes the subsequent direct phosphorylation of STAT3 by mTOR, which transactivates PDPN transcription.

    Journal: JCI Insight

    Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

    doi: 10.1172/jci.insight.186456

    Figure Lengend Snippet: ADAP is upregulated in sepsis and serves as an LPS stimulus–responsive protein that governs the upregulation of PDPN to form a distinct PDPN hi PM subset during sepsis, which displays a phenotype closely akin to M2 macrophages with enhanced phagocytic activity and provides enhanced protection against sepsis in the host. Mechanistically, while TLR4 signaling stimulates the upregulation of ADAP via the NF-κB pathway, TLR4-triggered phosphorylation of ADAP at Y 571 by BTK primes the subsequent direct phosphorylation of STAT3 by mTOR, which transactivates PDPN transcription.

    Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

    Techniques: Activity Assay