Journal: Life science alliance

Article Title: Antibodies targeting ADAM17 reverse neurite outgrowth inhibition by myelin-associated inhibitors.

doi: 10.26508/lsa.202403126

Figure Lengend Snippet: Figure 1. GT1b-mediated formation of the neuronal-receptor/co-receptor/transducer (NgR1, Lingo-1, p75) and the neuron-myelin (NgR1, Lingo-1, p75, MAG) signaling complexes. (A) Schematic representation of the neuronal receptor and co-receptor, the myelin inhibitor, and ADAM17 mediating the inhibitory signaling upon spinal cord injury. The colored parts represent the soluble, extracellular constructs that were used in this study and arrowheads denote the binding epitopes of the anti-ADAM17 mAbs. ICD denotes the intracellular domains. (B) Biochemical pull-down assay using protein A-Sepharose beads. Inputs: Lingo-Fc (10 μg); NgR1: (6 μg); p75: (4 μg); MAG: (8 μg). Where indicated, trisialoganglioside was added as a GT1b-Na salt. NgR1 and p75 were pre-incubated with GT1b-Na. Lingo-1-Fc was used as a “bait,” whereas the untagged NgR1, p75, and MAG proteins were used as “prey.” The protein A-Sepharose–bound fractions were analyzed by SDS–PAGE. (C) Gel Filtration (on a SD-200 column) profiles of Lingo-1, NgR1, and p75. The numbers above the gel lanes indicate the fraction numbers (each fraction is 1 ml). (D) Gel Filtration (on a SD-200 column) profiles of the NgR1, Lingo-1, p75 complex in the presence and absence of GT1b-Na. The elution volumes are listed above the peaks. The forward and reverse arrowheads denote the migration of NgR1 and p75, respectively, in the presence and absence of GT1b-Na salt. In the presence of GT1b-Na, all three proteins form a complex that elutes at 10.5 ml. The fractions from SD-200 were run on the SDS–PAGE. The numbers represent the different fractions from SD-200.

Article Snippet: Expression and purification of the protein constructs Ganglioside GT1b trisodium salt (CAS 59247-13-1) was purchased from Santa Cruz Biotechnology and used for the entire study.

Techniques: Construct, Binding Assay, Pull Down Assay, Incubation, SDS Page, Filtration, Migration

Journal: Life science alliance

Article Title: Antibodies targeting ADAM17 reverse neurite outgrowth inhibition by myelin-associated inhibitors.

doi: 10.26508/lsa.202403126

Figure Lengend Snippet: Figure 2. Interactions of ADAM17 and ADAM10 with the neuron-myelin signaling complex. (A) Purification of the 5-mer neuron-myelin signaling complex for ELISA-based studies. The 5-mer complex was formed in the presence of GT1b-Na as described in Fig 1. Briefly, bead-bound Lingo-1-Fc was used to pull-down NgR1, p75, and MAG in the presence of GT1b-Na. The beads were gently washed, and the bound proteins were eluted with low pH (100 mM glycine-HCl, pH 3.5). After elution, the pH was raised to 7 and the complex was analyzed on SDS–PAGE. (B, C) ELISA-based assay to gauge the binding of the 5-mer neuron-myelin complex (B) or p75 (C) to immobilized ADAM17 or ADAM10 (ECD or D+C protein constructs). (A) Briefly, wells were coated with ADAM17 ECD (E406A) or ADAM17 D+C or ADAM10 ECD (E384) or ADAM10 D+C, and varying concentrations of the 5-mer complex (isolated as described in panel (A)) or p75 were added to the wells and incubated for 1 h at RT. The bound p75 was detected by mouse mAb specific to the ectodomain of human p75. To detect the bound 5-mer complex, mouse mAb to the MAG (human) ectodomain was used. Data (n = 3) was recorded at 450 nm.

Article Snippet: Expression and purification of the protein constructs Ganglioside GT1b trisodium salt (CAS 59247-13-1) was purchased from Santa Cruz Biotechnology and used for the entire study.

Techniques: Enzyme-linked Immunosorbent Assay, SDS Page, Binding Assay, Construct, Isolation, Incubation

Journal: Life science alliance

Article Title: Antibodies targeting ADAM17 reverse neurite outgrowth inhibition by myelin-associated inhibitors.

doi: 10.26508/lsa.202403126

Figure Lengend Snippet: Figure 3. Biochemical pull-down experiments. (A) Binding of ADAM17 ECD (E406A) or ADAM17 D+C constructs to the neuronal receptor/co- receptor/p75 complex in the presence of GT1b-Na. Protein A-Sepharose pull-down assays were used to detect the binding of the ADAM17 ECD (active-site mutant E406A) (4 μg) or ADAM17 D+C (8 μg). The neuronal complex comprising Lingo-1- Fc, NgR1, and p75 was pre-formed in the presence of GT1b and used as a “bait.” The “prey” ADAM17 protein constructs were added, incubated, washed twice, and analyzed by SDS–PAGE. (B) Binding of ADAM17 ECD and ADAM17 D+C to the 5-mer neuron-myelin complex. Here, the pre-formed 5-mer complex was used as a “bait” to pull-down ADAM17 ECD or ADAM17 D+C. (C) Binding of ADAM17 D+C to isolated p75. The binding of ADAM17 D+C to Fc-tagged p75 was gauged using protein A-Sepharose beads with p75-Fc (4 μg) immobilized on the beads. The candidate ADAM17 D+C (8 μg) protein was added, washed, and bound proteins were detected by SDS–PAGE. (D) Binding of p75 to ADAM17 ECD. Because ADAM17 ECD and p75-Fc show identical mobility on SDS–PAGE (reducing conditions), we performed the experiment using untagged p75 (10 μg) as “prey” and ADAM17 ECD (E406A)-Fc (3 μg) immobilized on protein A-Sepharose beads.

Article Snippet: Expression and purification of the protein constructs Ganglioside GT1b trisodium salt (CAS 59247-13-1) was purchased from Santa Cruz Biotechnology and used for the entire study.

Techniques: Binding Assay, Construct, Mutagenesis, Incubation, SDS Page, Isolation

Journal: PLoS ONE

Article Title: Utilization of a Deoxynucleoside Diphosphate Substrate by HIV Reverse Transcriptase

doi: 10.1371/journal.pone.0002074

Figure Lengend Snippet: A. 5′ end-labeled primer, annealed to a template strand, was extended in the presence of a range of dADP concentrations by HIV-1 RT. The primer is extended by a single nucleotide, corresponding to the template ‘T’ at the +1 position. Extension reactions contained 10 nM wild-type enzyme, 5 nM primer-template, and were carried out for 10 minutes. Unextended primer is labeled ‘p’, the single nucleotide extension product is ‘p+1’. B. Single nucleotide extension assays of HIV-1 RT or KF exo- (50 nM each) with 10 µM of either dADP or dATP demonstrated no activity in the reaction containing KF exo- and dADP. Lane B, unextended primer. C. Single nucleotide extension assays utilizing dADP, dCDP, dGDP and dTDP incorporation opposite the complementary template nucleotide. Reactions were carried out with 100 µM of the appropriate dNDP or 0.2 µM dNTP, 50 nM enzyme and 5 nM template-primer. Lanes B1 and B2 contained RT and KF exo- respectively, with no nucleotide added. D. The RT preparation does not contain a contaminating nucleotide kinase activity. Nucleotides, which had been pre-incubated in the presence (labeled RT+ADP) or absence of HIV-1 RT, were used in a single nucleotide extension assay with either KF exo- or HIV-1 RT. Lane B: pre-incubation reaction contained no nucleotide. Unextended primer and primer extended by a single nucleotide are indicated ‘p’ and ‘p+1’ respectively.

Article Snippet: Nucleotides, oligonucleotides and commercial enzymes dADP, dCDP, dGDP, dTDP (dNDPs) and dAMP were purchased from MP Biomedicals.

Techniques: Labeling, Activity Assay, Incubation