quantikine elisa kit  (R&D Systems)

Journal: Nature Communications

Article Title: TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia

doi: 10.1038/s41467-024-48328-8

Figure Lengend Snippet: A GO term enrichment analysis of differentially expressed genes in F4/80 + CD169 + Vcam-1 + macrophages between TFPI f/f and TFPI f/f;EpoR mice. B Heatmap of differential gene expression. C mRNA expression of ALAS2, ALAD, HMBS, UROS, UROD, CPOX, and Fech in F4/80 + CD169 + Vcam-1 + macrophages of TFPI f/f;EpoR mice ( n = 5). D mRNA expression of ALAS2, ALAD, HMBS, UROS, UROD, CPOX, and Fech in F4/80 + CD169 + Vcam-1 + macrophages of rTFPI-treated mice ( n = 5). E Fech mRNA expression in F4/80 + CD169 + Vcam-1 + macrophages and Ter119 + cells of TFPI f/f;EpoR mice ( n = 5). (F and G) Heme content in F4/80 + CD169 + Vcam-1 + macrophages F and Ter119 + cells G of TFPI f/f;EpoR mice ( n = 5). H Heme content in Ter119 + cells of TFPI f/f;EpoR ;CD169 DTR/+ mice after DT treatment ( n = 5). I Heme content in Ter119 + cells of TFPI f/f;EpoR mice after clodronate liposomes treatment ( n = 5). J p-GATA1 and GATA1 protein levels in F4/80 + CD169 + Vcam-1 + macrophages of TFPI f/f;EpoR mice ( n = 3). K GATA1 protein expression of F4/80 + CD169 + Vcam-1 + macrophages after GATA1 shRNA treatment ( n = 3). L Heme content in F4/80 + CD169 + Vcam-1 + macrophages of TFPI f/f mice after treated with rTFPI and GATA1 shRNA ( n = 5). Statistical analysis was performed using one-way ANOVA ( A , and C – L ). Data are shown as mean ± SEM and are representative of two ( E – I ) or three (C, D, and J-L) independent experiments. Source data are provided as a Source Data file.

Article Snippet: Anti-mouse TFPI antibody (1:500, ab180619, Abcam, Cambridge, UK), anti-human TFPI antibody (1:500, ab260042, Abcam) anti-mouse Fech antibody (1:1000, 14466-1-AP, Proteintech, Wuhan, China), anti-mouse Thbd antibody (1:1000, ab230010, Abcam), anti-human Thbd antibody (1:1000, ab109189, Abcam), anti-mouse GATA1 antibody (1:1000, sc-265, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse PC antibody (1:1000, ab313386, Abcam), anti-Flag antibody (1:1000, F3165, Sigma-Aldrich), anti-Myc antibody (1:1000, 2276, Cell Signaling Technology), anti-GST antibody (1:1000, 2622, Cell Signaling Technology), anti-p-GATA1 antibody (1:500, PA5-104243, Thermo Fisher Scientific, Waltham, MA, USA), anti-β-actin antibody (1:1000, sc-47778, Santa Cruz Biotechnology) and anti-GAPDH antibody (1:1000, sc-365062, Santa Cruz Biotechnology).

Techniques: Expressing, Liposomes, shRNA

Journal: Nature Communications

Article Title: TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia

doi: 10.1038/s41467-024-48328-8

Figure Lengend Snippet: A mRNA expression of TF in F4/80 + CD169 + Vcam-1 + macrophages of Jak2 V617F -mutated or hypoxia-exposed mice ( n = 5). B TF procoagulant activity and plasma TAT levels in mice after TF mAb treatment ( n = 5). C Heme content in F4/80 + CD169 + Vcam-1 + macrophages of TFPI f/f;EpoR mice after TF mAb treatment ( n = 5). D Fech mRNA expression in F4/80 + CD169 + Vcam-1 + macrophages of TFPI f/f;EpoR mice after TF mAb treatment ( n = 5). E PB Hb in TFPI f/f;EpoR mice after TF mAb treatment ( n = 5). F Co-IP analysis of the interaction between TFPI and Thbd in HEK293T cells ( n = 2). G Schematic illustration of Thbd and TFPI constructs. H Co-IP analysis of the interaction of TFPI with the different domains of Thbd in HEK293T cells ( n = 2). I Co-IP analysis of the interaction between TFPI and Thbd in F4/80 + CD169 + Vcam-1 + macrophages ( n = 2). J Pull down analysis of the interaction between the TFPI and Thbd ( n = 2). K Thbd protein expression in F4/80 + CD169 + Vcam-1 + macrophages after Thbd shRNA treatment ( n = 3). L Fech mRNA expression in F4/80 + CD169 + Vcam-1 + macrophages after Thbd shRNA and rTFPI treatment ( n = 5). M Fech mRNA expression in HEK293T cells after Thbd transfection and rTFPI treatment ( n = 5). Statistical analysis was performed using one-way ANOVA ( B – E and K – M ). Data are shown as mean ± SEM and are representative of two ( B – J ) or three ( A and K – M ) independent experiments. Source data are provided as a Source Data file.

Article Snippet: Anti-mouse TFPI antibody (1:500, ab180619, Abcam, Cambridge, UK), anti-human TFPI antibody (1:500, ab260042, Abcam) anti-mouse Fech antibody (1:1000, 14466-1-AP, Proteintech, Wuhan, China), anti-mouse Thbd antibody (1:1000, ab230010, Abcam), anti-human Thbd antibody (1:1000, ab109189, Abcam), anti-mouse GATA1 antibody (1:1000, sc-265, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse PC antibody (1:1000, ab313386, Abcam), anti-Flag antibody (1:1000, F3165, Sigma-Aldrich), anti-Myc antibody (1:1000, 2276, Cell Signaling Technology), anti-GST antibody (1:1000, 2622, Cell Signaling Technology), anti-p-GATA1 antibody (1:500, PA5-104243, Thermo Fisher Scientific, Waltham, MA, USA), anti-β-actin antibody (1:1000, sc-47778, Santa Cruz Biotechnology) and anti-GAPDH antibody (1:1000, sc-365062, Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Co-Immunoprecipitation Assay, Construct, shRNA, Transfection

Journal: Nature Communications

Article Title: TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia

doi: 10.1038/s41467-024-48328-8

Figure Lengend Snippet: A mRNA expression of aPC in F4/80 + CD169 + Vcam-1 + macrophages of Jak2 V617F -mutated and hypoxia-exposed mice ( n = 5). B aPC protein expression of F4/80 + CD169 + Vcam-1 + macrophages after intraosseous infusion of lentivirus-expressing aPC shRNA ( n = 3). C Frequency of RIII, RIV, and RV erythroblast populations in Thbd f/f;LysM mice after intraosseous infusion of lentivirus-expressing aPC shRNA ( n = 5). D KEGG analysis of downregulated genes in F4/80 + CD169 + Vcam-1 + macrophages in TFPI f/f;EpoR mice. E Heme content in F4/80 + CD169 + Vcam-1 + macrophages treated with rTFPI combined with JNK, FOXO, and ERK1/2 inhibitors ( n = 5). F Heme content in F4/80 + CD169 + Vcam-1 + macrophages after treated with rTFPI combined with ERK1/2 inhibitor and GATA1 shRNA ( n = 10). G Phosphorylation level of ERK1/2 protein in F4/80 + CD169 + Vcam-1 + macrophages after rTFPI treatment ( n = 3). H Phosphorylation level of GATA1 protein in F4/80 + CD169 + Vcam-1 + macrophages of Thbd f/f;LysM mice after rTFPI treatment ( n = 3). I Phosphorylation level of GATA1 protein in F4/80 + CD169 + Vcam-1 + macrophages treated with rTFPI and ERK1/2 inhibitor ( n = 3). J Phosphorylation level of GATA1 protein in F4/80 + CD169 + Vcam-1 + macrophages of Thbd f/f;LysM mice at different time points after rTFPI treatment ( n = 3). K ALAS2 and Fech mRNA expression in F4/80 + CD169 + Vcam-1 + macrophages of Thbd f/f;LysM mice after rTFPI treatment ( n = 5). L ALAS2 and Fech mRNA expression in F4/80 + CD169 + Vcam-1 + macrophages after treated with rTFPI and ERK1/2 inhibitor ( n = 5). M ALAS2 and Fech mRNA expression in F4/80 + CD169 + Vcam-1 + macrophages after treated with rTFPI and GATA1 shRNA ( n = 5). Statistical analysis was performed using one-way ANOVA ( B , C , E , F , and K – M ). Data are shown as mean ± SEM and are representative of two ( C , E , and F ) or three ( A , B , and G – M ) independent experiments. N The signaling pathway of TFPI/Thbd mediated heme synthesis in F4/80 + CD169 + Vcam-1 + macrophages. Source data are provided as a Source Data file.

Article Snippet: Anti-mouse TFPI antibody (1:500, ab180619, Abcam, Cambridge, UK), anti-human TFPI antibody (1:500, ab260042, Abcam) anti-mouse Fech antibody (1:1000, 14466-1-AP, Proteintech, Wuhan, China), anti-mouse Thbd antibody (1:1000, ab230010, Abcam), anti-human Thbd antibody (1:1000, ab109189, Abcam), anti-mouse GATA1 antibody (1:1000, sc-265, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse PC antibody (1:1000, ab313386, Abcam), anti-Flag antibody (1:1000, F3165, Sigma-Aldrich), anti-Myc antibody (1:1000, 2276, Cell Signaling Technology), anti-GST antibody (1:1000, 2622, Cell Signaling Technology), anti-p-GATA1 antibody (1:500, PA5-104243, Thermo Fisher Scientific, Waltham, MA, USA), anti-β-actin antibody (1:1000, sc-47778, Santa Cruz Biotechnology) and anti-GAPDH antibody (1:1000, sc-365062, Santa Cruz Biotechnology).

Techniques: Expressing, shRNA

Journal: Nature Communications

Article Title: TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia

doi: 10.1038/s41467-024-48328-8

Figure Lengend Snippet: A Protocol used for human EBI formation. B TFPI expression in erythroblasts (EB) and macrophages (Ma) ( n = 3). C Thbd expression in erythroblasts and macrophages. Null means no expression ( n = 3). D hemoglobin and heme content in erythroblasts, heme content and Fech mRNA expression in macrophages of EBIs formed by macrophages and erythroblasts treated with TFPI shRNA ( n = 5). E hemoglobin and heme content in erythroblasts, heme content and Fech mRNA expression in macrophages of EBIs formed by macrophages and erythroblasts treated with TFPI shRNA and rTFPI ( n = 5). Statistical analysis was performed using one-way ANOVA ( B – E ). Data are shown as mean ± SEM and are representative of two (D and E) or three (B and C) independent experiments. Source data are provided as a Source Data file.

Article Snippet: Anti-mouse TFPI antibody (1:500, ab180619, Abcam, Cambridge, UK), anti-human TFPI antibody (1:500, ab260042, Abcam) anti-mouse Fech antibody (1:1000, 14466-1-AP, Proteintech, Wuhan, China), anti-mouse Thbd antibody (1:1000, ab230010, Abcam), anti-human Thbd antibody (1:1000, ab109189, Abcam), anti-mouse GATA1 antibody (1:1000, sc-265, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse PC antibody (1:1000, ab313386, Abcam), anti-Flag antibody (1:1000, F3165, Sigma-Aldrich), anti-Myc antibody (1:1000, 2276, Cell Signaling Technology), anti-GST antibody (1:1000, 2622, Cell Signaling Technology), anti-p-GATA1 antibody (1:500, PA5-104243, Thermo Fisher Scientific, Waltham, MA, USA), anti-β-actin antibody (1:1000, sc-47778, Santa Cruz Biotechnology) and anti-GAPDH antibody (1:1000, sc-365062, Santa Cruz Biotechnology).

Techniques: Expressing, shRNA