Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Nucleotide Sugar Transport in Trypanosoma brucei Alters Surface Glycosylation *

doi: 10.1074/jbc.M113.453597

Figure Lengend Snippet: TbNST4 is localized at the Golgi apparatus. A, genomic PCR analysis of in situ C-terminal HA-tagged tbnst4 in T. brucei BSF. Genomic DNA was isolated from untagged (lane 1) and tagged (lane 2) TbNST4 cells. In situ tagging locus was PCR amplified with a forward primer complementary to TbNST4 coding sequence and a reverse primer complementary to the 3′ UTR (outside the recombination sequence). The expected genomic PCR products are indicated. B, Western blot analysis of the in situ tagged TbNST4HA protein expression. Cell lysates from untagged (lane 1) and tagged TbNST4HA (lane 2) cells were subjected to SDS-PAGE and Western blotting with anti-HA antibody detection. The position of the expressed TbNST4 is indicated. C, immunofluorescence analysis of TbNST4HA localization. BSF cells carrying the tagged TbNST4 were stained with anti-HA for TbNST4 (green), anti-Ty for TbGT15 (red, upper panel), or anti-p67 (red, lower panel) antibodies. Cell images were captured with Nikon wide-field epifluorescence microscope. Blue, DAPI staining for nuclei and kinetoplasts.

Article Snippet: Cell images were captured with a Nikon wide-field epifluorescence microscope.

Techniques: In Situ, Isolation, Amplification, Sequencing, Western Blot, Expressing, SDS Page, Immunofluorescence, Staining, Microscopy

Journal:

Article Title: Heterogeneous Gene Expression and Functional Activity of Ryanodine Receptors in Resistance and Conduit Pulmonary as well as Mesenteric Artery Smooth Muscle Cells

doi: 10.1159/000127438

Figure Lengend Snippet: RyR subtypes are heterogeneously expressed in freshly isolated mouse RPA-SMCs, CPASMCs and MASMCs. a Expression of the smooth muscle-specific actin was found in nearly all isolated cells from resistance pulmonary arteries. Fluorescence image for actin staining (left) and transmitted light image (right) were simultaneously taken using a Zeiss LSM510 laser scanning confocal microscope. Cells were incubated with a primary antibody for smooth muscle-specific actin and then stained with Alexa Flour 488-conjugated anti-mouse antibody. b Gel electrophoresis reveals expression of RyR1, RyR2 and RyR3 mRNAs in RPASMCs, CPASMCs and MASMCs. The predicted PCR product sizes for RyR1, RyR2, RyR3 and GAP-DH were 81, 77, 83 and 122 bp, respectively. c Graphs show the relative mRNA expression levels of RyR1, RyR2 and RyR3 in RPASMCs, CPASMCs and MASMCs. The relative mRNA expression levels were obtained by normalizing the absolute expression levels of RyR1, RyR2 and RyR3 to that of GAPDH. Data were obtained from 6 separate experiments. * p < 0.05 compared with RyR1 in the same type of vascular SMCs; + p < 0.05 compared with RyR2 in the same type of cells; °p < 0.05 compared with RyR2 or RyR3 in RPASMCs.

Article Snippet: Measurements of [Ca 2+ ] i were made using a wide-field fluorescence microscope (IonOptix Corp., Milton, Mass., USA) [ 12 ].

Techniques: Isolation, Expressing, Fluorescence, Staining, Microscopy, Incubation, Nucleic Acid Electrophoresis

Journal: Nature Communications

Article Title: Distributed and retinotopically asymmetric processing of coherent motion in mouse visual cortex

doi: 10.1038/s41467-020-17283-5

Figure Lengend Snippet: a Schematic of the custom epifluorescent wide-field microscope for in vivo GCaMP6s imaging. The screen depicts a retinotopic mapping stimulus, with a drifting bar moving across the visual field (azimuth mapping). b Areal maps from one session of a single example mouse. Left: surface raw fluorescence image of a 4 mm cortical window of example Emx1-GCaMP6s mouse. Middle: horizontal and vertical retinotopic maps showing preferred location of each pixel for azimuth (left) and elevation (right); color bars indicate degree offset from center of visual field). Right: sign map (red, positive; blue, negative), and resulting segmentation of visual cortex into V1 and HVAs. Scale bars = 1 mm. c Schematic of the natural movie stimulus. Scenes were repeated 20 times to measure reliable neural responses. d Top: map from a single experiment showing reliability across posterior cortex; visual area segmentation as in ( b ). Bottom: multi-trial response (20 repeats) and mean trace (±s.e.m. shaded) of a single pixel (blue square) to repeated presentation of the natural movie. Reliability is defined as the across-trial Pearson correlation coefficient ( r = 0.31, see Methods). e Mean reliability map across all imaged mice ( n = 19 sessions over 7 mice). Individual maps are transformed onto a common coordinate system for comparison across mice. f Extraction of the motion energy in the stimulus. Pixel-wise motion vectors were extracted from each frame of the movie, and the sum of these vectors is used as a measure of the net motion energy of each frame. g Top: map from a single experiment showing motion response across posterior cortex; visual area segmentation as in ( b ). Bottom: Neural response of a single pixel (red) overlaid on the motion trace (gray); pixel-wise motion energy correlation is calculated as the Pearson correlation between these two signals ( r = 0.24). h Mean motion energy correlation map across all imaged mice ( n = 19 sessions over 7 mice); alignment procedure same as ( e ). Area abbreviations: primary visual (V1), lateral medial (LM), anterolateral (AL), posterior medial (PM), laterointermediate (LI), rostrolateral (RL), and anteromedial (AM).

Article Snippet: Fig. 1 Mesoscale calcium responses to motion energy in natural visual stimuli. a Schematic of the custom epifluorescent wide-field microscope for in vivo GCaMP6s imaging.

Techniques: Microscopy, In Vivo, Imaging, Fluorescence, Transformation Assay, Comparison, Extraction