American Society of Hematology

Figure 1

$Figure 1. HMW Angptl2 activates LILRB2 signaling. (A) Schematic of the chimeric LILRB2 receptor reporter system. (B) Representative flow cytometric profiles and summary showing that the Angptl2-conditioned medium stimulates GFP induction in the LILRB2 chimeric reporter system. The condition media of empty-vector–transfected HEK-293T cells was used as control. (C) Secreted Angplt2 and HLA-G-ECD in condition medium detected by anti-FLAG antibody in western blotting (left). Representative flow cytometric plots showing that Angptl2 binds to LILRB2 expressed on HEK293T cells better than the same amount of HLA-G-ECD (right). (D) The full-length (FL), CC, and FBN domains obtained from conditioned medium showed distinctive migration in reducing and nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis as determined by immunoblotting with anti-M2 Flag antibody. Protein extracted from equivalent amounts of condition media of empty-vector–transfected HEK293T cells was used as control. (E) GST-human Angptl2 purified from bacterial expression system by GST was immediately fractionated through gel-filtration FPLC. The molecular weight was determined by the peaks of apoferritin (443 kDa), amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa), respectively. (F) Equivalent amounts of indicated fractionated samples in FPLC were loaded on 10% native gel. Aggregated, monomeric, and cleaved GST-Angptl2 were visualized by silver staining. (G) Indicated FPLC fractionated samples were examined by western blotting using anti-M2 Flag antibody. The FLAG in cleaved GST-Angptl2 fragments (fraction 8; Figure 1G) could not be detected by western blotting. (H) Chimeric LILRB2 receptor reporter cells were treated with coated or soluble fraction 5 proteins for 48 hours. In coated wells, 5 µg/ml GST-Angptl2 from fraction 5 was precoated onto wells of a 96-well plate for 3 hours at 37°C. An equivalent amount of FPLC buffer was used as control. n.s. indicates not significant; ****P < .0001. Ctrl, control; KD, kilodalton.$

HMW Angptl2 activates LILRB2 signaling. (A) Schematic of the chimeric LILRB2 receptor reporter system. (B) Representative flow cytometric profiles and summary showing that the Angptl2-conditioned medium stimulates GFP induction in the LILRB2 chimeric reporter system. The condition media of empty-vector–transfected HEK-293T cells was used as control. (C) Secreted Angplt2 and HLA-G-ECD in condition medium detected by anti-FLAG antibody in western blotting (left). Representative flow cytometric plots showing that Angptl2 binds to LILRB2 expressed on HEK293T cells better than the same amount of HLA-G-ECD (right). (D) The full-length (FL), CC, and FBN domains obtained from conditioned medium showed distinctive migration in reducing and nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis as determined by immunoblotting with anti-M2 Flag antibody. Protein extracted from equivalent amounts of condition media of empty-vector–transfected HEK293T cells was used as control. (E) GST-human Angptl2 purified from bacterial expression system by GST was immediately fractionated through gel-filtration FPLC. The molecular weight was determined by the peaks of apoferritin (443 kDa), amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa), respectively. (F) Equivalent amounts of indicated fractionated samples in FPLC were loaded on 10% native gel. Aggregated, monomeric, and cleaved GST-Angptl2 were visualized by silver staining. (G) Indicated FPLC fractionated samples were examined by western blotting using anti-M2 Flag antibody. The FLAG in cleaved GST-Angptl2 fragments (fraction 8; Figure 1G) could not be detected by western blotting. (H) Chimeric LILRB2 receptor reporter cells were treated with coated or soluble fraction 5 proteins for 48 hours. In coated wells, 5 µg/ml GST-Angptl2 from fraction 5 was precoated onto wells of a 96-well plate for 3 hours at 37°C. An equivalent amount of FPLC buffer was used as control. n.s. indicates not significant; ****P < .0001. Ctrl, control; KD, kilodalton.

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