Journal:

Article Title: Biochemical and Genetic Characterizations of a Novel Human Immunodeficiency Virus Type 1 Inhibitor That Blocks gp120-CD4 Interactions

doi: 10.1128/JVI.77.19.10528-10536.2003

Figure Lengend Snippet: Competitive inhibition of gp120-CD4 binding by BMS-378806 and binding affinity of BMS-378806 to gp120. (A) Saturation binding curve of BMS-378806. The levels of sCD4 bound to gp120 in the absence or presence of various concentrations of BMS-378806 and in escalating concentrations of sCD4 were measured by using the gp120-CD4 binding ELISA. BMS-378806 concentrations: ▪, 0 μM; ▴, 0.8 μM; ▾, 1.6 μM; ⧫, 3.2 μM. Data were analyzed according to the one-site binding model with GraphPad Prism. OD450, optical density at 450 nm. (B) Apparent binding constants for sCD4 in the absence and presence of BMS-378806. The Kd and Bmax values were derived by using nonlinear regression analysis with GraphPad Prism. Values were the means ± standard errors of the means, representing two independent experiments. (C) Binding affinity of BMS-378806 to gp120 by SPA. [3H]BMS-378806 binding to the gp120 protein was measured by employing gp120-coated SPA beads. The amount of gp120-bound [3H]BMS-378806 was determined by using a Packard TopCount scintillation counter. The results from Scatchard analysis of binding isotherm are shown in the insert. The x axis represents bound (corrected), and the y axis indicates bound/free.

Article Snippet: The curve represents the best fit to a saturation binding isotherm determined by using GraphPad Prism.

Techniques: Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal:

Article Title: Biochemical and Genetic Characterizations of a Novel Human Immunodeficiency Virus Type 1 Inhibitor That Blocks gp120-CD4 Interactions

doi: 10.1128/JVI.77.19.10528-10536.2003

Figure Lengend Snippet: Binding stoichiometry of BMS-378806 to gp120 protein. gp120JRFL (1.0 μM) was titrated with BMS-378806, and the observed percent reduction in fluorescence from each of four independent experiments was plotted first as a function of BMS-378806 concentration (data not shown). Each of these data sets was then normalized to the percent maximal fluorescence reduction observed for each experiment in order to analyze the four sets of data together. The individual, normalized data sets are depicted as open squares, closed squares, closed diamonds, and open triangles. The curve represents the best fit to a saturation binding isotherm determined by using GraphPad Prism.

Article Snippet: The curve represents the best fit to a saturation binding isotherm determined by using GraphPad Prism.

Techniques: Binding Assay, Fluorescence, Concentration Assay

Journal: Cell

Article Title: LAP2 Proteins Chaperone GLI1 Movement Between Lamina and Chromatin to Regulate Transcription

doi: 10.1016/j.cell.2018.10.054

Figure Lengend Snippet: (A) BASU-GLI1 vicinal labeling in ASZ followed by streptavidin pulldown ± CRT0329868 (CRT) (+biotin, +CRT, 5hr). (B) APEX2-GLI1 vicinal labeling in ASZ followed by streptavidin pulldown ± PSI. (C) Co-IP of FLAG-GLI1 in ASZ ± PSI followed by immunoblot. (D) PLA between total GLI1 and LAP2α (top) or LAP2β (bottom) in ASZ treated with indicated inhibitors for 2hr (scale bar=20μm, n=10 fields, ANOVA). (E) PLA between total GLI1 and LAP2α (left) or LAP2β (right) in 1º human BCCs treated with vorinostat ex vivo (scale bar= 66μm, n=10 fields, ANOVA). (F) Co-IP of in vitro translated HA-GLI1 zinc-finger domain (HA-GLI1ZF) from WCE. Inputs in Figure S5B. (G) LAP2-binding mutants mapped onto GLI1:DNA crystal structure (pdb:2GLI). Mutations which inhibit (red) or are permissive of (grey) LAP2 binding are illustrated as spheres. Co-IP in Figure S5C. (H) Co-IP of HA-GLI1WT/T296E transfected into HEK293T followed by immunoblot of endogenous LAP2. Inputs in Figure S5D. (I) qRT-PCR of GLI1 and GAPDH following transfection of GLI1WT/T296E into NIH3T3 (n=9, ANOVA). Associated immunoblot in Figure S5E. (J) Co-IP of full length GLI1 (GLI1 FL) or zinc-finger domain GLI1 (GLI1 ZF) with recombinant LAP2 constant region (−/+ indicate the addition of LAP2 peptide). Input in Figure S5F. (K) Co-IP of wheat germ cell extract in vitro translated HA-GLI1 with chemically synthesized biotin-LEM-like (residues 5–48), biotin-LEM (residues 109–153), or biotin-scrambled LEM-like domains. Associated inputs and saturation binding experiment in Figure S5G and S5H. (L) Co-IP of FLAG-GLI1 co-transfected into HEK293T with a gradient of LAP2α, followed by immunoblot for total LAP2 (n=3). Input and reciprocal IP in Figures S5I and S5J. (M) GLI1 transfected in HEK293T (top, cellular IP) or in vitro translated and incubated in WCE (bottom three, in vitro IP) with indicated mutations/truncations co-IP with associated epitope tag. IP washed over a gradient of high salt conditions prior to immunoblot. Complex strength=−slope(x)−1−slope(α)−1 Error bars represent standard error, error bars omitted when smaller than the width of associated data point symbol, ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S5.

Article Snippet: Curve fitting for FRAP analysis and saturation binding was also performed in GraphPad PRISM 6.

Techniques: Labeling, Co-Immunoprecipitation Assay, Western Blot, Ex Vivo, In Vitro, Binding Assay, Transfection, Quantitative RT-PCR, Recombinant, Synthesized, Incubation

Journal: Bioconjugate chemistry

Article Title: Generation of clickable Pittsburgh Compound B for the detection and capture of β-amyloid in Alzheimer’s Disease brain

doi: 10.1021/acs.bioconjchem.7b00500

Figure Lengend Snippet: (A) A saturation binding assay was used to derive a Kd of 164.7 nM for fluorescent clicked PiB (4). (B) Dose-response curve showing displacement of fluorescent clicked PiB (4) (at 140 nM) with increasing concentrations (0–100,000 nM) of PiB (2) ( ) and clickable PiB (3) ( ). From these data, binding affinities (Ki) of 678.4 and 264.7 nM were determined for compounds 2 and 3, respectively (summarized in Table 1).

Article Snippet: Binding affinities were derived using the saturation binding one-site specific non-linear regression analysis model of GraphPad Prism 5 (GraphPad Software Inc., California).

Techniques: Saturation Assay, Binding Assay