Journal: The Journal of Neuroscience

Article Title: In Vivo Assessment of Brain Interstitial Fluid with Microdialysis Reveals Plaque-Associated Changes in Amyloid-β Metabolism and Half-Life

doi: 10.1523/JNEUROSCI.23-26-08844.2003

Figure Lengend Snippet: In vitro microdialysis to measure Aβ. A, Diagram of exchangeable Aβ. Triangles represent potential Aβ binding molecules, e.g., apoE, clusterin, and α2M. Only highlighted Aβ molecules are of the appropriate size to pass through a 35 kDA MWCO membrane on the microdialysis probe. B, Interpolated zero flow method to quantify the pool of measurable Aβ1-x and Aβ40 within samples of human CSF (n = 4). At 2.2 μl/min, the percentage recovery of eAβ was 9.74 ± 1.53% (mean ± SEM). C, In vitro percentage recoveries for each Aβ species using the interpolated zero flow method. Each recovery point contains error bars and are overlapping for each species (n = 4). In vitro recovery of each Aβ species by microdialysis is the same. D, The concentration of eAβ and total soluble Aβ are highly correlated within a sample of human CSF (Pearson's r = 0.9487; p < 0.0001; n = 9). The mean concentrations of soluble Aβ1-x and eAβ1-x were 30.47 ± 4.23 ng/ml (mean ± SEM) and 196.2 ± 28.65pg/ml, respectively. E, F, Human CSF immunoprecipitated (IP'd) for Aβ has undetectable levels of total soluble Aβ and eAβ. Human CSF spiked with an amount of exogenous Aβ40 peptide expected to double Aβ concentration resulted in a 2.1-fold increase in total soluble Aβ and a 1.9-fold increase in eAβ.

Article Snippet: Microdialysis probes used for in vitro experiments had a 4 mm, 35 kDa molecular weight cutoff (MWCO) membrane (BR-4-10 probes; Bioanalytical Systems, West Lafayette, IN) and were connected to a microdialysis peristaltic pump (MAB20; SciPro, Sanborn, NY) using teflon (FEP) tubing (inner diameter, 0.12 mm; 1.2 μl/10 cm; SciPro).

Techniques: In Vitro, Binding Assay, Membrane, Concentration Assay, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Mitotic checkpoint gene expression is tuned by codon usage bias

doi: 10.15252/embj.2021107896

Figure Lengend Snippet: A Cellular protein noise (coefficient of variation, CV = std / mean) in live‐cell microscopy images of S. pombe ; n = 7 images (Nmt1‐GFP), 11 (Mad1‐GFP), 19 (Mad2‐GFP), 10 (Mad3‐GFP); single images had 16–79 GFP‐positive and 6–94 GFP‐negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all P < 0.001). B Simulations of stochastic gene expression noise from selected mRNA/protein half‐life combinations assuming a constantly active promoter (see Methods). Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6,000 per cell. The x‐axis of each graph shows time, the y‐axis shows mRNA number per cell (blue) or protein number per cell (black). C Theoretical prediction for the coefficient of variation (CV = std/mean) of the protein number per cell, assuming different mRNA and protein half‐lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6,000 (approx. 100 nM). D mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for noninstantaneous labeling by 4tU. Act1 + and ecm33 + were used as long and short half‐life controls, respectively; qPCR was performed for the endogenous mRNAs. Half‐lives (95% confidence interval): mad1 + 5.6 min (4.3–8.4), mad2 + 7.7 min (6.2–10.4), mad3 + 5.2 min (4.3–6.9), act1 + 61.8 min (37.2–172.3), ecm33 + 5.0 min (4.5–5.7). E Protein abundances after translation shut‐off with cycloheximide (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one‐phase exponential decay. Cdc2 and Cdc13 were used as long and short half‐life controls, respectively. Immunoblots for the endogenous proteins (no tag). A representative experiment shown in Appendix Fig . Source data are available online for this figure.

Article Snippet: Cycloheximide (from Streptomyces griseus) , Chem Impex , Cat # 00083.

Techniques: Microscopy, Negative Control, Expressing, Labeling, Western Blot

Journal: The EMBO Journal

Article Title: Mitotic checkpoint gene expression is tuned by codon usage bias

doi: 10.15252/embj.2021107896

Figure Lengend Snippet:

Article Snippet: Cycloheximide (from Streptomyces griseus) , Chem Impex , Cat # 00083.

Techniques: Recombinant, In Vitro, Sequencing, Labeling, Protease Inhibitor, Isolation, Magnetic Beads, Bicinchoninic Acid Protein Assay, Software, Membrane