differentiation 36  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

doi: 10.1073/pnas.2104166118

Figure Lengend Snippet: Fig. 4. C1s inhibits binding of IT4var20 IE to EPCR and IT4var13 IE binding to CD36 but not ICAM-1. (A) Binding of IT4var20 IEs to EPCR in the absence or presence of C1s (100 μg/mL). Recombinant EPCR (rEPCR) was included as a control and indicator of how well C1s treatment abrogated IT4var20 binding to EPCR. (B) Binding of IT4var13 IEs to CD36 or ICAM-1 in the absence or presence of C1s (100 μg/mL). In both binding assays, bovine serum albumin (BSA) was included as a negative control. The mean and SEM are shown, and each data point is from an independent experiment. For statistical testing, all mean values were log-transformed, and a two-tailed unpaired t test was conducted to compare experimental groups to the positive control (EPCR only). (C) Var gene transcript levels relative to the housekeeping control gene, seryl-t-transferase, confirming the major var types being expressed as IT4var20 and IT4var13. *P < 0.05; **P < 0.01; ns, not significant.

Article Snippet: Plastic plates were spotted with 5 μL of EPCR (13320-H02H; Sino Biologicals), CSA (a gift from Dr. Michal Fried, NIAID/NIH, Bethesda, MD), CD36 (1955-CD; R&D Systems), or ICAM-1 (720-IC; R&D Systems); all recombinant proteins, except for ICAM-1 which was used at 100 μg/mL, were at a concentration of 50 μg/mL.

Techniques: Binding Assay, Recombinant, Control, Negative Control, Transformation Assay, Two Tailed Test, Positive Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

doi: 10.1073/pnas.2104166118

Figure Lengend Snippet: Fig. 6. Analysis of C1s-treated recombinant PfEMP1. (A) SDS/PAGE gel showing two representative PfEMP1 CIDR domains (EPCR-binding IT4var19 CIDRα1 and CD36-binding CIDRα3) and two EPCR-binding head-structure domain complexes (PFD1235w [NTSA-DBLα1-CIDRα1] and IT4var20 [NTSA-DBLα1-CIDRα1]) before and after treatment with 50 nM C1s at 1:1 molar ratio for 1 h at 37 °C. No proteolysis was seen. (B) ELISA data showing the effect of 50 nM C1s pretreatment on binding of recombinant PfEMP1 proteins to coated EPCR, CD36, or ICAM-1 (filled boxes) versus EPCR binding to coated recombinant PfEMP1 (hashed boxes). Whereas C1s treatment reduced binding activity of full-length PfEMP1 ectodomains, recombinant PfEMP1 head structure domains are insensitive to C1s treatment, indicating that C1s cleavage sites are located in the interdomain regions, consistent with MS analysis. As a control, C1s treatment of EPCR did not affect its binding to full-length PfEMP1 ectodomains (hashed boxes).

Article Snippet: Plastic plates were spotted with 5 μL of EPCR (13320-H02H; Sino Biologicals), CSA (a gift from Dr. Michal Fried, NIAID/NIH, Bethesda, MD), CD36 (1955-CD; R&D Systems), or ICAM-1 (720-IC; R&D Systems); all recombinant proteins, except for ICAM-1 which was used at 100 μg/mL, were at a concentration of 50 μg/mL.

Techniques: Recombinant, SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Control