Journal: bioRxiv

Article Title: Axonal ER tubules regulate local translation via P180/RRBP1-mediated ribosome interactions

doi: 10.1101/2022.11.30.518484

Figure Lengend Snippet: (A, D, G) Representative STED (A), TREx (D) and dual-color SMLM (G) images of the ER and ribosomes in axons from neurons expressing GFP-Sec61β and stained for RpS12. (B, E, H) Magnifications and intensity profile lines from merged images for each microscopy method. (C, F, I) Quantification of RpS12 intensity in ER mask, enlarged ER mask, and one-color flipped images, for each microscopy method. (J) Representative STED images and intensity profile line for an axon segment of a neuron transfected as in (A) and co-labeled for puromycilated peptides. (K) Quantification of RpS12 intensity in ER mask and enlarged ER mask with or without high puromycin treatment using dual-color SMLM. (L-N) Schematic representation of split APEX system used to detect ER-ribosome contacts. When the ER protein Sec61β fused to AP module and the ribosomal protein RpL10A fused to EX module interact with each other, APEX is reconstituted and contact sites can be visualized as a biotinylation radius around the interactions (L). Representative images of split APEX assay in distal axons from neurons expressing RpL10A-3xHA-EX and V5-AP-Sec61β (left), or V5-AP-RTN4A as a negative control (right). Expression of constructs are visualized with V5 and HA antibodies, and biotinylation is detected with conjugated Strep-555 (M). Quantification of Strep signal in distal axons from neurons as in (M), and without H2O2 as a negative control for the biotinylation reaction (N). (O) Quantification of axonal ER-bound ribosomes using split APEX assay with RpL10A-3xHA-EX and V5-AP-Sec61β, in neurons stimulated for 30-minutes with BSA (control), BDNF, NT-3 or NGF. Individual data points each represent a neuron in (C, F, I, K, N and O). Boxplots show 25/75-percentiles, the median, and whiskers represent min to max in (C, F, I and K). Data are presented as mean values ± SEM in (N, O). ns = not significant, *p < 0.05, **p < 0.01, ***p<0.001 comparing conditions to control using unpaired t-tests or ordinary one-way ANOVA tests. Scale bars represent 1μm (A, D, G, J) and 5μm (M).

Article Snippet: Other reagents used in this study were: Puromycin dihydrochloride (Sigma-Aldrich, Cat# P8833), Anisomycin (Sigma-Aldrich, Cat#9789), recombinant human Neurotrophin-3 (NT-3) protein (50ng/ml, Alomone labs, Cat#N-260), recombinant human BDNF protein (50ng/ml, Alomone labs, Cat#B-250), recombinant rat beta-NGF Protein (50ng/ml, R&D systems, Cat# 556-NG).

Techniques: Expressing, Staining, Microscopy, Transfection, Labeling, Negative Control, Construct

Journal: Bioengineered

Article Title: Interleukin-17A attenuates photoreceptor cell apoptosis in streptozotocin-induced diabetic mouse model

doi: 10.1080/21655979.2022.2084241

Figure Lengend Snippet: The lack of IL-17A blocks high glucose-induced mature NT-3 production . (a, b) Representative immunoblots and densitometric quantitation of proNT-3 and mature NT-3 protein amounts in MMC isolated from IL-17A −/− and WT mice, under NG or HG conditions for 18 hours. (c) Representative immunoblots and densitometric quantitation of proNT-3 and mature NT-3 protein amounts in MMC pretreated with various amounts of recombinant IL-17A (0, 10, 50, 100 and 200 ng/mL) for 18 hours. Data are mean ± SEM (n = 3 independent samples t test, **P < 0.01, ***P < 0.001 compared to 0 ng/mL group). Note: MMC, Müller cell; HG, high glucose; NG, normal glucose; WT, wild type.

Article Snippet: This was followed by overnight incubation at 4°C with anti-IL-17A (1/200, Abcam, Cambridge, MA, USA), anti-glutamine synthetase (GS; 1/1000, Abcam), anti-GFAP (1/10 000, Abcam), anti-Bcl-2 (1/1000, Abcam), anti-Bax (1/2000, Abcam), anti-active caspase-3 (1/500, Abcam), anti-NT-3 (1/500, Alomone Labs, Jerusalem, Israel), anti-pro-NT3 (1/500, Alomone Labs), anti-TrkC (1/2000, Abcam), anti-P75 NTR (1/2000, Abcam), and anti-β-actin (1/5000; Cell Signaling Technology, Beverly, MA, USA).

Techniques: Western Blot, Quantitation Assay, Isolation, Recombinant