mouse anti βiii tubulin  (R&D Systems)

Journal: Life Science Alliance

Article Title: Locally synthesized glycyl aminoacyl-tRNA synthetase is important for local translation in neurons

doi: 10.26508/lsa.202603630

Figure Lengend Snippet: N2a cells were transfected with antisense oligos (ASO) or a scrambled-sequence control (Scrmbl), or were kept untransfected (−), then differentiated for 24 h. (A) Cells were subjected to RNA extraction followed by RT–qPCR to measure levels of tRNA Gly . ΔC t was calculated by subtracting the C t mean of tRNA Gly from that of β-actin. Each triangle indicates a biological replicate ( n = 3 biological replicates). (B) Cells were fixed to visualize the transfection efficiency. Transfection efficiency was calculated by dividing Cy5-positive cells to the total number of cells ( n = 68 and 74 cells in scrambled and ASO groups, respectively, from three biological replicates; ∼22–28 cells per replicate). Scale bar, 40 μm. (C) Cells were subjected to immunofluorescence to assess the morphology of neurons after transfection of ASOs and a scrambled control. Neurites are stained with βIII-Tub (orange), nuclei with Hoechst (cyan). Scale bar, 20 μm. (A, B) Data information: P -values: (NS) > 0.05 by the Kruskal–Wallis test (A) and Pearson’s chi-squared test (B). (B) Results are presented as the mean ± SEM (B).

Article Snippet: βIII-tubulin , Developmental Studies Hybridoma Bank (DSHB) , 6G7 , 1:200.

Techniques: Transfection, Sequencing, Control, RNA Extraction, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: PLOS One

Article Title: Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress

doi: 10.1371/journal.pone.0345890

Figure Lengend Snippet: (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and β3-tubulin (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.

Article Snippet: Primary antibodies for immunofluorescence include mouse anti-βIII Tubulin (R&D Systems, MAB1195), rabbit anti-AQP4 (Millipore Sigma, HPA014784), chicken anti-GFP (Aves Labs, Inc., GFP-1020), rabbit anti-p62 (Abcam, Ab109012 ), mouse anti-Ubiquitin (Enzo Life Sciences, ENZ-ABS840; − and ), mouse anti-Ubiquitin (Enzo Life Sciences, BML-PW8810; , and ), and rat anti-LAMP1 (Abcam, ab25245).

Techniques: Transgenic Assay, Marker, Labeling, Solvent, Control, Ubiquitin Proteomics