Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic lupus erythematosus immune complexes increase the expression of SLAM family members CD319 (CRACC) and CD229 (LY-9) on plasmacytoid dendritic cells and CD319 on CD56(dim) NK cells.

doi: 10.4049/jimmunol.1301022

Figure Lengend Snippet: FIGURE 5. pDCs lack the activating adaptor molecules but express inhibitory adaptor molecules of SLAM receptors. (A) IFN-a production by RNA-IC–stimulated pDCs after cross-linking of CD319, CD229, or BDCA-2 by plate-bound mAbs. IFN-a levels were determined in 20-h cell culture supernatants by an immunoassay and normalized to the IFN-a production in cultures without plate-bound mAb. The dashed line indicates 100%. Data (mean + SD) are from three donors from two independent experiments. Expression of EAT2 and SAP (B) and SHIP-1, SHP-1, SHP- 2, and CSK (C) in cell lysates from isolated pDCs or NK cells from two healthy donors was determined by Western blot. Expression of b-actin was used as control.

Article Snippet: Rabbit polyclonal Abs to Ewing’s sarcoma-activated transcript 2 (EAT2), SHIP-1, SHP-2, CSK (Proteintech Group, Chicago, IL), and a rabbit mAb to SHP-1 (EPR5519; Epitomics, Burlingame, CA), followed by HRP-conjugated goat anti-rabbit IgG (H+L) (Invitrogen), were used to detect the indicated signaling molecules.

Techniques: Cell Culture, Expressing, Isolation, Western Blot, Control

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

Article Title: Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection

doi: 10.1073/pnas.1610197114

Figure Lengend Snippet: Schematics of the anti-HCV drug targets and the experimental system. (A) HCV life cycle and drug targets. After entry into the host cell, HCV genomic RNA is translated into viral precursor polyprotein and processed into functional proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). HCV RNA replicates inside the isolated membrane compartments derived from the endoplasmic reticulum (ER), and assembles into viral particles on lipid droplets, which traffic through the Golgi and are released outside of the cell. PIs (TPV, DPV, ASV, and SMV) inhibit the processing step, and drugs such as NI (SOF), NNIs (VX, DAS, NSV, and TGV), NS5AIs (DCV and LDV), and CIs (CsA and SCY) target HCV RNA replication. IFNs (IFN-α and IFN-λ1) supposedly inhibit at least the step(s) of translation and replication. (B) HCV replication activity was evaluated using an HCV subgenomic replicon (genotype 1b, strain NN) carrying a fusion of the firefly luciferase gene (Luc) with the neomycin phosphotransferase (Neor). The replicon autonomously and persistently replicates in Huh-7 cells. Cells treated with drugs were incubated for 72 h and then harvested for luciferase assay. Inhibition of HCV replication was measured by the luciferase activity in drug-treated cells, relative to activity in DMSO-treated cells.

Article Snippet: SMV, ASV, DAS, NSV, TGV, and LDV were purchased from MedChem Express.

Techniques: Functional Assay, Isolation, Membrane, Derivative Assay, Activity Assay, Luciferase, Incubation, Inhibition

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

Article Title: Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection

doi: 10.1073/pnas.1610197114

Figure Lengend Snippet: Quantification of the IIP of single-HCV drugs. (A) Log–log plots of dose–response curves normalized by IC50, determined from the replicon assay, of PIs (TPV, DPV, SMV, and ASV; red), the NI (SOF; blue), NNIs (VX, DAS, NSV, and TGV; orange), NS5AIs (DCV and LDV; green), IFNs (IFN-α and IFN-λ1; cyan), and CIs (CsA and CSY; purple). Each point represents the mean of three experiments. (B) IIP of classes or subclasses of antiviral drugs, normalized by IC50, calculated from the experimentally measured fu by Eq. 1. (C) IIP values at drug concentration D = 100 × IC50 (IIP100) determined by extrapolation.

Article Snippet: SMV, ASV, DAS, NSV, TGV, and LDV were purchased from MedChem Express.

Techniques: Concentration Assay

Journal: Science Advances

Article Title: A phospho-tyrosine–based signaling module using SPOP, CSK, and LYN controls TLR-induced IRF activity

doi: 10.1126/sciadv.abq0084

Figure Lengend Snippet: ( A and B ) IP/IB analysis of HEK293T cells based on exogenously expressed proteins. The SBM in CSK and its mutation in CSK-AAA are indicated. ( C ) FA competition binding assay. Peptides containing the candidate SBM of CSK and the mutated sequence CSK-AAA compete with fluorescein-Puc 91–106 for binding to the SPOP MATH domain. [L] is the peptide concentration. Continuous lines are nonlinear least-squares fits to a complete competitive binding model. See for K d values. ( D ) Clustal Omega–based sequence alignment of Src family kinases and CSK. The SBM and the conserved tyrosine are highlighted by green and red boxes, respectively. ( E ) IP/IB analysis of HEK293T cells based on exogenously expressed proteins. ( F ) Nuclear translocation assay of SPOP-deficient RAW264.7 cells that were reconstituted with indicated forms of SPOP. ( G ) qPCR analysis of cells described in (F) that were stimulated with CpG-DNA for 4 hours. ( H to K ) In vitro kinase assays based on proteins expressed in HEK293T cells. CSK-AAA, SST340/341/341AAA. Fold differences of CSK autophosphorylation as determined by phosphor image analysis. The activity used for reference (set as 1) is underlined. Data represent mean ± SD from three independent experiments. ** P < 0.01, *** P < 0.005 are determined by two-way ANOVA with Sidak’s multiple comparison test (G).

Article Snippet: N-terminal FLAG-tagged mouse CSK-WT and CSK-SST340/341/342AAA were expressed using the vector pCMV3-N-FLAG (Sino Biological). pcDNA3-N-FLAG-hIRF8 was from Sino Biological.

Techniques: Mutagenesis, Binding Assay, Sequencing, Concentration Assay, Nuclear Translocation Assay, In Vitro, Activity Assay