Journal: Molecular & Cellular Proteomics

Article Title: Accurate Quantification of Cardiovascular Biomarkers in Serum Using Protein Standard Absolute Quantification (PSAQ™) and Selected Reaction Monitoring

doi: 10.1074/mcp.m111.008235

Figure Lengend Snippet: FIG. 3. PSAQ-SRM quantification of cardiovascular biomarkers in patient samples and correlation with enzymatic or ELISA as- says. A, Correlation between total CK enzymatic activity and CKMB concentration, as determined by PSAQ-SRM, in clinical serum

Article Snippet: ELISA and Enzymatic Assays—Myoglobin concentration in patient serum samples was determined using the Human Myoglobin ELISA Kit (Alpco Diagnostics, Salem, NH) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Concentration Assay

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

Article Title: Amyloid precursor protein and C99 are subunits in human microglial Hv1 channels that enhance current and inflammatory mediator release

doi: 10.1073/pnas.2509903122

Figure Lengend Snippet: APP knockdown reduces Hv1 currents in human iMG. iMG were studied by whole-cell patch clamp using a holding voltage of −60 mV, 0.5 s test pulses, and a 10 s interpulse interval, with pH i = 6.0 and pH o = 7.5. Values are mean ± SEM; n = 5-9 cells for each condition. ( A ) Representative proton current traces in the absence (−C6) and presence (+C6) of 2 μM C6 peptide, a specific inhibitor of Hv1 channels. Inset : voltage-step protocol consisting of 20 mV increments from −60 mV to +60 mV. ( B ) Peak proton current densities at 0 mV in the absence (−C6) or presence (+C6) of 2 μM C6 peptide. ( C ) Dose–response relationships for C6 inhibition of proton current at 0 mV. K i for C6 was estimated to be 297 ± 7 nM from a Hill equation fit (h = 1.6 ± 0.1). ( D ) Conductance–voltage relationships (G-V) for proton currents in iMG and cloned Hv1 expressed in HEK293T cells (HEK). ( E ) Representative proton currents in iMG treated with Scr siRNA as a negative control and APP siRNA knockdown (20 mV steps from −60 to +60 mV). APP knockdown slowed current activation 2.9-fold (4,100 ± 550 ms vs. 11,800 ± 1,700 ms) and speeded deactivation 1.7-fold (310 ± 40 ms vs. 180 ± 30 ms) at 0 mV. ( F ) Peak current densities of proton currents at 0 mV for iMG treated with Scr siRNA or with APP siRNA knockdown. ( G ) G-V relationships for proton currents in iMG treated with Scr siRNA or APP siRNA. APP knockdown induces a significant depolarizing shift (+9 mV, P = 0.01) in the V 1/2 of the G-V relationship.

Article Snippet: APP knockdown was achieved using the APP Human siRNA Oligo Duplex (OriGene), following the manufacturer’s protocol.

Techniques: Knockdown, Patch Clamp, Inhibition, Clone Assay, Negative Control, Activation Assay

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

Article Title: Amyloid precursor protein and C99 are subunits in human microglial Hv1 channels that enhance current and inflammatory mediator release

doi: 10.1073/pnas.2509903122

Figure Lengend Snippet: APP knockdown reduces release of cytokines and ROS from iMG. Cytokines were quantified using ELISA as described in Materials and Methods . Values are mean ± SEM; n = 6 for each condition. Control (CTL) represents no LPS stimulation ( A ) C6 (2 μM) treatment decreased LPS-stimulated release of TNF-α, IL-6, IL-1β, IFN-γ, IL-2, IL-4, IL-8, IL-10, IL-12p70, and IL-13 concentrations. **** P ≤ 0.0001. ( B ) TNF-α release in response to LPS in the absence or presence of 2 μM C6 peptide or KTx* peptide (a negative control peptide that does not block ion channels) from iMG treated with Scr siRNA or APP siRNA knockdown. ( C ) IL-6 release in response to LPS in the absence or presence of 2 μM C6 or KTx* peptide from iMG treated with Scr siRNA or APP siRNA knockdown. ( D ) ROS release in response to LPS in the absence or presence of 2 μM C6 or KTx* peptide from iMG treated with Scr siRNA or APP siRNA knockdown.

Article Snippet: APP knockdown was achieved using the APP Human siRNA Oligo Duplex (OriGene), following the manufacturer’s protocol.

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Control, Negative Control, Blocking Assay