Journal: Frontiers in Pharmacology

Article Title: Pharmacological Insights Into Safety and Efficacy Determinants for the Development of Adenosine Receptor Biased Agonists in the Treatment of Heart Failure

doi: 10.3389/fphar.2021.628060

Figure Lengend Snippet: Multi-pathway profiling at the A 2A R, A 2B R and A 3 R. Adenosine receptor agonism was analyzed in CHO-hA 2A R (left panels), CHO-hA 2B R (center panels) and CHO-hA 3 R cells (right panels) . Cells were analyzed in cAMP (top row) , calcium (second row) , and phosphorylation of ERK1/2 (third row) and Akt1/2/3 (fourth row) , with calculated bias shown in the lower row. As bias data are normalized to NECA and the cAMP pathway, where there is no response to NECA at a pathway, ligand-induced cAMP in a cell line, bias cannot be calculated. Statistical analyses of bias are shown in . Concentration response data are expressed as mean ± SEM.

Article Snippet: For concentration response data, curves were analyzed using three-parameter nonlinear curve fitting (GraphPad Prism 8.02) of grouped data. pK b values of VCP746 at hA 3 receptor were determined using the Schild method ( ) and calculated using the operational model plus agonism (GraphPad Prism 8.02).

Techniques: Phospho-proteomics, Concentration Assay

Journal: The Journal of Immunology Author Choice

Article Title: Fc Galactosylation Promotes Hexamerization of Human IgG1, Leading to Enhanced Classical Complement Activation

doi: 10.4049/jimmunol.2100399

Figure Lengend Snippet: IgG glycoengineering for altered galactosylation and sialylation. (A) Schematic setup for the production process in HEK FreeStyle cells, with the addition of relevant substrates and constructs coding for enzymes prior/during transfection. (B and C) Fc glycosylation profiles of produced anti-biotin mAbs using different glycoengineering techniques to increase Fc galactosylation and sialylation, analyzed by mass spectrometry. The bar graphs represent the mean and SEM of five different mAbs. For the statistical analysis, an ordinary one-way ANOVA with Tukey multicomparison test was performed. (D–F) Relative levels of IgG binding of produced anti-biotin mAbs to 5× biotin/BSA are presented as relative value to the maximum response of the unmodified WT mAb, determined by ELISA (n = 3). Curve fitting was performed using nonlinear regression dose-response curves with log(agonist) versus response–variable slope (four parameters) in GraphPad Prism 8.0.2. No differences in opsonization were observed between glycovariants. **p ≤ 0.01, ****p ≤ 0.0001.

Article Snippet: Data represent the mean and SEM of three independent experiments; curve fitting was performed using nonlinear regression dose-response curves with log(agonist) versus response–variable slope (four parameters) in GraphPad Prism 8.0.2. ( I ) The maximum response and ( J ) EC 50 of complement-mediated lysis of glycoengineered and mutated anti-biotin mAbs extracted from ( .

Techniques: Construct, Transfection, Produced, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Fc Galactosylation Promotes Hexamerization of Human IgG1, Leading to Enhanced Classical Complement Activation

doi: 10.4049/jimmunol.2100399

Figure Lengend Snippet: Complement-mediated lysis of biotinylated Raji cells. (A) Flow cytometric gating strategy; cells were gated based on the forward-/side-scatter, and the percentage of dead cells was calculated using the LIVE/DEAD Fixable Near-IR Dead Cell Stain. (B) Complement-mediated lysis with unmodified anti-biotin variants. (C–H) Complement-mediated lysis using glycoengineered anti-biotin variants. Data represent the mean and SEM of three independent experiments; curve fitting was performed using nonlinear regression dose-response curves with log(agonist) versus response–variable slope (four parameters) in GraphPad Prism 8.0.2. (I) The maximum response and (J) EC50 of complement-mediated lysis of glycoengineered and mutated anti-biotin mAbs extracted from (Fig. 5C–H. For the statistical analysis, an ordinary one-way ANOVA with Tukey multicomparison test was performed. Nonsignificant comparisons were not shown. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

Article Snippet: Data represent the mean and SEM of three independent experiments; curve fitting was performed using nonlinear regression dose-response curves with log(agonist) versus response–variable slope (four parameters) in GraphPad Prism 8.0.2. ( I ) The maximum response and ( J ) EC 50 of complement-mediated lysis of glycoengineered and mutated anti-biotin mAbs extracted from ( .

Techniques: Lysis, Staining