Evaluation of the potency and selectivity of APP blocker-9. Sanghamitra Bandyopadhyay Catherine Cahill Amelie Balleidier Conan Huang Debomoy K. Lahiri Xudong Huang Jack T. Rogers 10.1371/journal.pone.0065978.g004 https://plos.figshare.com/articles/figure/_Evaluation_of_the_potency_and_selectivity_of_APP_blocker_9_/760764 <p><b>Panel A</b>: Dose responsive measurement of the capacity of JTR-009 to limit APP 5′UTR-luciferase expression relative to posiphen, a known APP translation blocker (JTR-009: IC<sub>50</sub> = 0.1 µM; posiphen: IC<sub>50</sub> = 5 µM, N = 4). <b>Panel B</b>: Dose-responsive reduction APP levels in SH-SY5Y cells treated 48 hours at 0.1 µM, 0.5 µM and 1 µM JTR-009. Western blot for APP levels using N- terminal 22C11 antibody (standardization with β-actin as loading control). Bottom Panel: Histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells. <b>Panel C</b>: Lysates from the experiment in Panel B was analyzed by Western blotting using APP the C-terminal specific (A8717) antibody and β-actin antibody. Bottom Panel: histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells from autoradiographic film subjected to densitometry (N = 3). <b>Panel D</b>: Dose-responsive capacity of JTR-009 to limit APP expression in primary E-18 mouse neurons (1 nM). The relative α-synuclein (<i>SNCA</i>) expression was calculated. Shown, the combined data was graphed into a histogram where mean values from separate assays were calculated from densitometry of Western blots (N = 5). <b>Panel E</b>: Real-time qPCR measurement of the dose-responsive measurement of the levels of APP mRNA in SH-SY5Y cells treated with escalating concentrations of JTR-009 for 48 hours. <b>Panel F</b>: Equivalent real-time qRT-PCR analysis to measure APP mRNA and TfR mRNA levels in SH-SY5Y cells after 48 h treatment with 25 µM desferrioxamine (DFO) (Positive control for qRT-PCR analysis shown in Panel E).</p> 2013-07-31 01:51:32 Biochemistry Nucleic acids rna proteins genetics Molecular cell biology Cellular types neurons gene expression Protein translation nanotechnology ferritin Clinical genetics Chromosomal disorders Down syndrome neurology dementia Alzheimer disease potency selectivity app