Journal: bioRxiv

Article Title: Myosin-II mediated traction forces evoke localized Piezo1 Ca 2+ flickers

doi: 10.1101/294611

Figure Lengend Snippet: A. Schematic of the Molecular Tension Sensor (MTS) used for traction force imaging. The N-terminal region of the sensor (blue) is tethered to a PEG-functionalized glass coverslip. A fibronectin domain at the C-terminal end (brown) binds to the cell’s integrins (purple), allowing cells to attach to the glass coverslip. An elastic spring domain bridges the two ends of the sensor and separates a FRET donor (green) and acceptor (red). Cell-generated traction forces pull the FRET pair apart, resulting in reduced FRET efficiency. The FRET index (the ratio of acceptor intensity over summed donor and acceptor intensities), serves as a measure of force; with a low FRET index indicating high force, and a high FRET index indicating a low force. B. Imaging of Piezo1 Ca 2+ flickers. Panels show resting fluorescence of HFF cells loaded with Ca 2+ indicator Cal-520, with overlaid red dots marking the centroid locations of Ca 2+ flickers. C. Corresponding force maps from the same cells, overlaid with red dots marking the Ca 2+ flicker locations. Blue denotes low FRET (high force) and red denotes high FRET (low force). The color bar in C represents FRET efficiency (top) and the average force per MTS per pixel in pN obtained from calibrated FRET-Force curves of the MTS as described in Methods. D. Box and whisker plots with red boxes showing distances from Piezo1 flicker localizations to the nearest traction force region for HFFs (top: 515 flickers from 9 cells) and hNSPCs (bottom: 66 flickers from 18 cells). Grey boxes show corresponding mean distances derived from simulations of 9000 random intracellular locations for each cell. Box range is 25 th to 75 th percentile; whiskers denote 10 th and 90 th percentile, horizontal lines represent the median and filled black squares represent mean. *** denotes p < 0.001 by Kolmogorov-Smirnov test.

Article Snippet: Mat-tek dishes of shRNA-transfected cells loaded with Cal-520 AM were first scanned using an Olympus UPLSAPO 10× objective to identify cells expressing TurboRFP.

Techniques: Imaging, Generated, Fluorescence, Whisker Assay, Derivative Assay

Journal: Frontiers in Neurology

Article Title: Biochemical Distribution of Tau Protein in Synaptosomal Fraction of Transgenic Mice Expressing Human P301L Tau

doi: 10.3389/fneur.2014.00026

Figure Lengend Snippet: Soluble and sarkosyl-insoluble tau in JNPL3 male mice . (A) Western blots of TBS-soluble tau in mouse cerebral cortices. Equal volumes of TBS-soluble fraction derived from 0.2 mg wet weight of brain from eight male JNPL3 and two male non-tg mice were separated by SDS-PAGE, blotted, and then probed with E1, Tau5, MS06, and GAPDH antibodies. (B) Western blot of sarkosyl-insoluble fractions in mouse cerebral cortices. Samples derived from 20 mg wet weight from male JNPL3 and non-tg mice, 5 mg wet weight from female JNPL3 cortex (ctx), and 2.5 mg wet weight from female JNPL3 spinal cord (SPc) were separated by SDS-PAGE, blotted, and then probed with E1 antibody.

Article Snippet: The samples were separated by gel electrophoresis on 10 or 5–20% gradient SDS-PAGE gels (Wako Pure Chemical Industries, Osaka, Japan), and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany).

Techniques: Western Blot, Derivative Assay, SDS Page

Journal: Frontiers in Neurology

Article Title: Biochemical Distribution of Tau Protein in Synaptosomal Fraction of Transgenic Mice Expressing Human P301L Tau

doi: 10.3389/fneur.2014.00026

Figure Lengend Snippet: Subcellular fractionation of mouse cerebral cortex . (A) Schematic representation of the subcellular fractionation steps. P1, nuclear pellet and debris; P2, crude synaptosomal fraction; P3, light membranes; S3, cytosolic fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction; LS2, soluble synaptosomal fraction. (B) Western blots of JNPL3 and non-tg male mouse cerebral cortex subcellular fractions. P1, P2, P3, or S3 fraction derived from 0.13 mg wet weight of tissue and LP1, LP2, or LS2 fraction derived from 0.5 mg wet weight of tissue were loaded on SDS-PAGE. Blots were probed with PSD-95, synaptotagmin, GAP-43, Tau5, E1, β-tubulin, and β-actin antibodies. (C) Proportions of protein levels in fractions (P1, P2, P3, and S3) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice are shown. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (P1 + P2 + P3 + S3). Results are expressed as mean ± SEM. (D) Proportions of protein levels in synaptosomal fractions (LP1, LP2, and LS2) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice were indicated. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (LP1 + LP2 + LS2). Results are expressed as mean ± SEM.

Article Snippet: The samples were separated by gel electrophoresis on 10 or 5–20% gradient SDS-PAGE gels (Wako Pure Chemical Industries, Osaka, Japan), and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany).

Techniques: Fractionation, Western Blot, Derivative Assay, SDS Page, Imaging

Journal: Frontiers in Neurology

Article Title: Biochemical Distribution of Tau Protein in Synaptosomal Fraction of Transgenic Mice Expressing Human P301L Tau

doi: 10.3389/fneur.2014.00026

Figure Lengend Snippet: Quantitative western blot analysis of tau protein . (A) Tau band patterns in cytosolic (S3) and synaptosomal membrane (LP1) fractions from four JNPL3 and four non-tg mouse cerebral cortices. Equal volumes of fractions derived from 0.25 mg wet weight of brain were separated by SDS-PAGE, blotted, and then probed with Tau5, MS06, Tau1, pS199, pT231, and pS396 antibodies. (B,C) The relative ratio of tau protein between S3 and LP1 fractions from JNPL3 (B) and non-tg (C) mice was measured ( n = 4 each). Results are expressed as mean ± SEM. The mean value of tau protein in S3 fraction from JNPL3 mice was normalized to one.

Article Snippet: The samples were separated by gel electrophoresis on 10 or 5–20% gradient SDS-PAGE gels (Wako Pure Chemical Industries, Osaka, Japan), and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany).

Techniques: Western Blot, Derivative Assay, SDS Page