Journal: PLoS ONE

Article Title: Tissue plasminogen activator mediates deleterious complement cascade activation in stroke

doi: 10.1371/journal.pone.0180822

Figure Lengend Snippet: (A) C3 protein was incubated with plasmin. Plasmin-mediated C3a production increased in a dose-dependent manner. ***P<0.001 vs. control (n = 3). (B) tPA promotes C3 cleavage in human plasma, and this process is suppressed by α2-antiplasmin but not by CD35. *P<0.05, **P<0.01, ***P<0.001 vs. controls (n = 3). (C) Following intravenous tPA infusion in C57BL/6 mice in the absence of ischemia, a significant increase in plasma C3a levels was observed. *P<0.05 (n = 3).

Article Snippet: To assess the mechanism by which tPA cleaves C3 in plasma ex vivo , we incubated tPA (100μg/ml) with human plasma (1:1000, Sigma-Aldrich) with and without α-2 anti-plasmin (Abcam; 100nM) or recombinant CD35 (R&D systems; 20nM) for 90 minutes at 37 ° C. C3a concentration was determined using ELISA.

Techniques: Incubation, Control, Clinical Proteomics

Journal: International Journal of Nanomedicine

Article Title: An Immunoreceptor-Targeting Strategy with Minimalistic C3b Peptide Fusion Enhances SARS-CoV-2 RBD mRNA Vaccine Immunogenicity

doi: 10.2147/IJN.S463546

Figure Lengend Snippet: Confirmation of targeting proficiency for mC3 and mFc ligands via immunofluorescence assay (IFA). ( A ) Illustration of the ligands-receptors interactions that were demonstrated by IFA. Following mRNA transfection into 293T cells, receptors were incubated with the cells. Then, RBD-mC3 bound CR1 while RBD-mFc bound FcγR. The RBD-mC3 and RBD-mFc were labeled by anti-RBD monoclonal antibodies. The CR1 and FcγR were marked using anti-CR1 polyclonal antibodies and anti-FcγR monoclonal antibodies, respectively. ( B ) Antibodies targeting CR1 (FITC, green) and RBD-mC3 (Cy5, purple) depicted the respective distributions of CR1 and RBD-mC3. ( C ) Colocalization analysis of RBD-mC3 and CR1. ( D ) Antibodies against FcγR (green) and RBD-mFc (purple) showed the distributions of FcγR and RBD-mFc respectively. The blue color (DAPI) represented the cell nuclei. ( E ) Colocalization analysis of RBD-mFc and FcγR. Three regions of interest (ROI) were analyzed. Pearson’s coefficient (R) was calculated in colocalization analysis. R > 0.8 suggests a very strong correlation.

Article Snippet: Then, the cells were blocked with 10% goat serum (Jackson ImmunoResearch) for 1 hr at room temperature, followed by one-hour incubation of receptor proteins, including recombinant human CD35 protein (CR1) (R&D Systems #5748-CD-050, NE Minneapolis, MN) and mouse CD64/FCGR1 protein (Sino Biological #50086-M08H).

Techniques: Immunofluorescence, Transfection, Incubation, Labeling, Bioprocessing