Journal: Biochimica et Biophysica Acta

Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

doi: 10.1016/j.bbamcr.2013.02.012

Figure Lengend Snippet: Immunocytochemical distribution of mCherry-RTN1 523 and RyRs in HEK293 cells. (A) HEK293 cells were transiently transfected with mCherry-RTN1 523 in the absence (a) or presence of untagged RyR2 (d–f) or RyR1 (g–i). Single-transfections of untagged RyR2 (b) and RyR1 (c) were carried out as a comparison. Cells were immunostained with anti-RyR2 (b,d–f) or anti-RyR1 (c,g-i) antibodies and visualized by confocal microscopy. Note that single-transfected mCherry-RTN1 523 is uniformly distributed in the cytosol (a), but altered to a more reticular staining pattern when co-expressed with RyR2 (d–f). mCherry-RTN1 523 remained uniformly distributed in cells cotransfected with RyR1 (g–i). Cells shown represent at least 20 representative cells per condition. (B) Immunofluorescence confocal microscopy analysis of RyR ER localization in HEK293 cells. HEK293 cells were single-transfected with untagged RyR1 cDNA or RyR2 cDNA, immunostained with the indicated antibodies and imaged using immunofluorescence microscopy to demonstrate RyR and calreticulin (ER-specific marker) colocalization. Cells were immunostained for RyR isoforms (green) and Calreticulin (red), respectively. Space bar: 10 μm. (C) Extent of colocalization between mCherry-RTN1 523 and RyR isoforms was quantified using Pearson correlation coefficient and determined through correlation analysis with Leica SP5 software from 10 different cells per group. (*** p = 0.003 by Student's t -test).

Article Snippet: Single cells transfected with untagged RyR1 or RyR2 were double stained with rabbit anti-calreticulin (1:500; Abcam) and mouse pan-RyR antibody (1:1000; Pierce).

Techniques: Transfection, Confocal Microscopy, Staining, Immunofluorescence, Microscopy, Marker, Software

Journal: Diabetes

Article Title: Functional Role of Neuroendocrine-Specific Protein-Like 1 in Membrane Translocation of GLUT4

doi: 10.2337/db09-0756

Figure Lengend Snippet: Distributions of GLUT4 in the transverse tubules. A–D : Comparison of the distribution of GLUT4 near the transverse tubules recognized by anti-GLUT4 (red) and anti–ryanodine receptor (green) in the wild-type ( A and B ) or sk-NSPl1–deficient ( C and D ) muscles, without ( A and C ) or with ( B and D ) electrical stimulation for 15 min. Ryanodine receptor was located very close to the transverse tubules, and thus it was used as the transverse-tubule membrane or A-I junction marker. GLUT4 is located on the transverse tubules or A-I junctions, as seen by the intensity of yellow ( B and C ). E : Typical quantification of GLUT4 translocation in the transverse tubules. After obtaining the surrounding image, the confocal image at the ryanodine receptor–abundant section was again scanned (12 μm × 12 μm, 2048 × 2048 pixels) ( A–D ). From the recorded image, distances between two A-I junctions were measured (i.e., the white dashed line shown in A and the box illustrated in E , 200–350 pixels/line), judging from ryanodine receptor immunofluorescence (green circles), and then normalized (0 and 1.0, horizontal axis in F and G ). Intensities of GLUT4 immunofluorescence along this line (red circles) were also measured, and the means at every 0.06 normalized distance along the line were plotted against the normalized distance of the A-I junction. F and G : Results of the GLUT4 translocation in the transverse tubules. Black and red dashed lines represent the normalized distance, where the intensity from GLUT4 reached a peak value without or with stimulation, respectively. Wild type: without stimulation, n = 60 (couples of transverse tubules from two mice); with stimulation, n = 71 (three mice). sk-NSPl1 deficient: without stimulation, n = 67 (two mice); with stimulation, n = 55 (two mice). Bar = 1 μm. E-stim, electrical stimulation; RyR, ryanodine receptor. (A high-quality color digital representation of this figure is available in the online issue.)

Article Snippet: The thin muscles were fixed by 3.5% freshly depolymerized paraformaldehyde for 2 h. After washing with Tris-buffered saline (TBS; pH 7.4), single muscles were treated with 0.05% Triton X-100 for 15 min and then with 5% skim milk for 15 min. Then the muscles were incubated in TBS containing several primary antibodies (anti-GLUT4 and -dystrophin [Chemicon] and anti–ryanodine receptor [Affinity BioReagents]) overnight.

Techniques: Marker, Translocation Assay, Immunofluorescence