Journal: The Journal of International Medical Research

Article Title: Icariin influences cardiac remodeling following myocardial infarction by regulating the CD147/MMP-9 pathway

doi: 10.1177/0300060518762060

Figure Lengend Snippet: Expression levels of CD147, MMP-9, and TIMP-1 of rat hearts in different groups. (a) mRNA expression levels. (b) Protein expression levels. (d) Activity of MMP-9. * P

Article Snippet: The rats in the model group were randomly divided into 3 groups of 10 rats each and injected with 2 mL/kg per day of physiological saline, 12 mg/kg per day of icariin (Sigma-Aldrich), and 12 mg/kg per day of icariin + 10 mg/kg per day of CD147 (Sino Biological Inc.), respectively.

Techniques: Expressing, Activity Assay

Journal: The Journal of Cell Biology

Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

doi: 10.1083/jcb.201801081

Figure Lengend Snippet: The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and β-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.

Article Snippet: MYC-ACVR2A and CER1-FLAG plasmids were purchased from Sino Biological (MG50613-NM and MG51161-CF, respectively).

Techniques: Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay

Journal: The Journal of Cell Biology

Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development

doi: 10.1083/jcb.201801081

Figure Lengend Snippet: AMOP domain is required for the function of ISM1 as an antagonist of NODAL signaling. (A) Diagram of domain-deleted ISM1 constructs lacking either TSR1 or AMOP (ΔTSR1 and ΔAMOP, respectively). The N -glycosylation sites at positions N39 and N282 are indicated. N39 is located in the N terminus, 10 amino acids downstream from the signal peptide (amino acids 1–29), and N282 is located in the region between TSR1 and AMOP domains (amino acids 260–285). (B) Western blot of ISM1 in WCL and CM samples of HEK293T cells transiently transfected with wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1. Samples were digested with PNGase F or Endo H before being subjected to Western blotting. (C) Mapping of NODAL-interacting domain of ISM1. HEK293T cells were transiently transfected with FLAG-NODAL together with either wild-type ISM1 or domain-deleted ISM1 mutants. CM samples were immunoprecipitated (IP) with anti-FLAG or anti-ISM1 antibodies and subjected to Western blotting with anti-ISM1 and anti-FLAG antibodies. (D) Mapping of ACVR1B ECD -interacting domain of ISM1. HEK293T cells transiently expressing ACVR1B ECD were cotransfected with wild-type ISM1 or domain-deleted ISM1 mutants. ACVR2B ECD were also cotransfected with ACVR1B ECD to potentially improve the interaction between ISM1 and ACVR1B ECD . CM samples were immunoprecipitated with anti-ISM1 antibodies and immunoblotted with anti-HA antibodies. ISM1 interaction with ACVR1B ECD is no longer observed when AMOP domain is absent. (E) Western blot of pSMAD2 in WCL of P19C6 cells treated with 100 ng/ml NODAL in the presence of mock, HIS-CER1, FLAG-LEFTY1, wild-type ISM1, ΔTSR1-ISM1, or ΔAMOP-ISM1 CM. Western blots for HIS (CER1), FLAG (LEFTY1), and ISM1 in CM samples is shown.

Article Snippet: MYC-ACVR2A and CER1-FLAG plasmids were purchased from Sino Biological (MG50613-NM and MG51161-CF, respectively).

Techniques: Construct, Western Blot, Transfection, Immunoprecipitation, Expressing

Journal: Frontiers in Immunology

Article Title: Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

doi: 10.3389/fimmu.2019.02920

Figure Lengend Snippet: Characterization of the isolated FcγRIV-specific VHH. (A) The predicted amino acid residue sequence of FcγRIV-VHH-7m, isolated after panning on recombinant mouse FcγRIV protein of a VHH library derived from a llama immunized with immature mouse dendritic cells. Above the sequences the Kabat numbering is indicated. CDR1, −2, and −3 are boxed. (B) Flow cytometric analysis showing binding of FcgRIV-VHH-7m and M2e-VHH-23m to HEK 293T cells transiently transfected with expression vectors coding for mouse FcγRI, FγRIIb, FcγRIII, and FcγRIV along with the common γ-chain for the activating FcγRs and a GFP expression plasmid. The lower graph depicts binding to Mf4/4 cells. The cells were incubated with fourfold serial dilution of Alexa Fluor™ 647 labeled FcγRIV-VHH-7m or M2e-VHH-23m, starting at a concentration of 0.2 μM, binding of the VHHs to the GFP positive cells was analyzed. Data points represent averages of triplicates and error bars standard deviations.

Article Snippet: VHH Binding to FcγRs Expressing CellsHuman Embryonic Kidney (HEK) 293T cells were transiently transfected with full length mouse FcγRI (MG50086-CF), FcγRIIb (MG50030-CY, SinoBiological Inc.), FcγRIII (MG50326, SinoBiological Inc.) or FcγRIV (MG50036-CF, SinoBiological Inc.) expression constructs along with the common γ-chain for the activating FcγRs (MG50935-CF) by polyethylenimine (PEI)-based transfection.

Techniques: Isolation, Sequencing, Recombinant, Derivative Assay, Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Serial Dilution, Labeling, Concentration Assay