p150glued  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8 + T lymphocytes

doi: 10.3389/fimmu.2024.1411957

Figure Lengend Snippet: Glycolytic response during T-cell receptor (TCR) activation requires active cytosolic protein translation in human CD8 + cytotoxic T lymphocytes. (A) Glycolytic response estimated by extracellular acidification rate (ECAR) in resting or stimulated human cytotoxic T lymphocytes (CTLs) treated with vehicle (CTRL), cycloheximide (CHX; 20 μg/mL), or puromycin (PURO; 50 μg/mL) for 1 h. Stimulation was with αCD3/αCD28 tetramers. Glucose, oligomycin, and 2-deoxyglucose (2-DG) were injected as indicated. (B, C) Graphs show the rate of (B) glycolysis and (C) glycolytic capacity. Data are mean ± SEM from five technical replicates from six healthy donors. (D) Oxygen consumption rate (OCR) from the experiment in panel (A) . (A, D) A representative experiment out of six. Linear mixed model was used to analyse differences in (A) ECAR and (D) OCR and one-way ANOVA test for (B) glycolysis and (C) glycolytic capacity. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, non-significant. (E) Phosphorylation of Akt (S473), mTOR (S2448), and S6 (S235/S236) in CTLs pre-treated as in panel A and stimulated with αCD3/αCD28 tetramers at indicated times. Graphs showing quantification of densitometries of (F) Akt (S473), (G) mTOR (S2448), and (H) S6 (S235/S236) blots; data are normalised to p150-Glued and referenced to non-stimulated CTRL. Data are mean ± SEM; (F) , n = 4 (G) , n = 5 (H) , n = 4; two-way ANOVA. *, p < 0.05; **, p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Anti-CD3ϵ (clone HIT3a), anti-CD28 (clone CD28.2), anti-p150-Glued [1/p150Glued (RUO); 1:1,000 WB], anti-CD8-V500 (1:100 FACS), and anti-CD14-PE (1:100 FACS) were from BD Biosciences (San Jose, CA, USA).

Techniques: Activation Assay, Injection

Journal: Frontiers in Immunology

Article Title: Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8 + T lymphocytes

doi: 10.3389/fimmu.2024.1411957

Figure Lengend Snippet: Inhibition of cytosolic protein synthesis during T-cell receptor (TCR) and CD28 stimulation modulates the recruitment of tubulin molecular motors to mitochondria in human CD8 + cytotoxic T lymphocytes. (A, B) Western blotting showing distribution of components of dynein (p74-dynein intermediate chain) and kinesin-1 [Kinesin heavy chain (KHC); kinesin light chain (KLC)] molecular motors and the dynactin adaptor (p150-Glued), K40-acetylated and total α-tubulin, and β-actin and VDAC in (A) total lysate and (B) isolated mitochondria from cytotoxic T lymphocytes (CTLs) treated with vehicle (CTRL), cycloheximide (CHX; 20 μg/mL), or puromycin (PURO; 50 μg/mL) for 1 h and stimulated with αCD3/αCD28 tetramers for 15 min. A representative experiment out of four is shown. Graphs showing densitometric quantification ratios of indicated proteins normalised to VDAC and referenced to non-stimulated CTRL. Box and whiskers, data are median plus min and max from n = 4 healthy donors; two way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant.

Article Snippet: Anti-CD3ϵ (clone HIT3a), anti-CD28 (clone CD28.2), anti-p150-Glued [1/p150Glued (RUO); 1:1,000 WB], anti-CD8-V500 (1:100 FACS), and anti-CD14-PE (1:100 FACS) were from BD Biosciences (San Jose, CA, USA).

Techniques: Inhibition, Western Blot, Isolation

Journal: Journal of Virology

Article Title: Herpes simplex virus type-1 cVAC formation in neuronal cells is mediated by dynein motor function and glycoprotein retrieval from the plasma membrane

doi: 10.1128/jvi.00713-24

Figure Lengend Snippet: Localization of DIC and p150Glued in infected CAD cells. ( A and B ) Digital confocal images of mock-infected CAD cells that were immunofluorescently stained for indicated proteins are shown. The arrowhead in ( A ) indicates the MTOC. ( C–E ) CAD cells infected with HSV at m.o.i. = 5 for 12 h before fixation and staining are shown. In ( C ), arrowheads indicate the position of the cVAC, where pUL11 and p150Glued co-localize. The white dotted outline indicates the boundary of the nucleus defined by Hoechst staining. In ( D and E ), cyan and yellow arrowheads indicate landmark p150Glued puncta outside and inside the cVAC, respectively. The cyan line in ( D ) indicates the line along which the Z-sections shown in ( E ) were taken. Scale bars in A–C represent 10 µm. Scale bar for D and E represents 4 µm.

Article Snippet: The primary antibodies were diluted as follows: rabbit anti-HA epitope tag antibodies (Abcam) 1:500, rabbit anti-UL51 antiserum (gift from Joel Baines at Cornell University) 1:500, rabbit anti-UL11 antiserum (gift from John Wills at Penn State University) 1:1,500, mouse monoclonal anti-gE antibody (Virusys) 1:500, goat anti-p150Glued antibody (Abcam) 1:500, mouse anti-DIC antibody (Santa Cruz Biotechnology) 1:250, mouse anti-GM130 antibody (BD Bioscience), and 1:500 mouse anti-ß-tubulin antibody (Sigma-Aldrich).

Techniques: Infection, Staining

Journal: Journal of Virology

Article Title: Herpes simplex virus type-1 cVAC formation in neuronal cells is mediated by dynein motor function and glycoprotein retrieval from the plasma membrane

doi: 10.1128/jvi.00713-24

Figure Lengend Snippet: Localization of viral structural components relative to p150Glued. Digital images of CAD cells infected at m.o.i. = 5 for 12 h are shown. ( A–C ) CAD cells infected with HSV(F) ( A and B ) or HSV-1 35R ( C ). In ( B ), arrows indicate puncta outside the cVAC, where pUL11, p150Glued, and gE co-localize. In ( C ), white arrows indicate p150Glued puncta outside the cVAC that did not contain detectable capsid signal, while yellow arrows indicate p150Glued puncta outside the cVAC that co-localizes with capsids. All scale bars represent 10 µm.

Article Snippet: The primary antibodies were diluted as follows: rabbit anti-HA epitope tag antibodies (Abcam) 1:500, rabbit anti-UL51 antiserum (gift from Joel Baines at Cornell University) 1:500, rabbit anti-UL11 antiserum (gift from John Wills at Penn State University) 1:1,500, mouse monoclonal anti-gE antibody (Virusys) 1:500, goat anti-p150Glued antibody (Abcam) 1:500, mouse anti-DIC antibody (Santa Cruz Biotechnology) 1:250, mouse anti-GM130 antibody (BD Bioscience), and 1:500 mouse anti-ß-tubulin antibody (Sigma-Aldrich).

Techniques: Infection

Journal: Acta Neuropathologica Communications

Article Title: Axonal autophagic vesicle transport in the rat optic nerve in vivo under normal conditions and during acute axonal degeneration

doi: 10.1186/s40478-024-01791-2

Figure Lengend Snippet: Changes of autophagic pathways and motor proteins in the rat optic nerve 6 h after crush lesion. A Representative immunoblots of LC3, p62, Kif5, Dynein, p150Glued, Cathepsin D, and GAPDH 6 h after crush injury in the areas of the optic nerve 1 mm proximal and distal to the lesion site compared with the contralateral side. B – H Quantifications of the band intensities of LC3 II:LC3 I, p62, Kif5, Dynein, p150Glued, and Cathepsin D normalized to GAPDH as loading control. Error bars represent Mean ± SEM. 3 independent experiments were included. *P < 0.05; **P < 0.01 N.S. no significant difference, according to one-way ANOVA and Tukey multiple comparisons test

Article Snippet: Antigen retrieval was performed in cell conditioning solution (pH 8.5) at 50 ℃ for 4 h. After blocking in Dako diluent reagent for 1 h at room temperature, sections were incubated with the following primary antibodies: anti-p-ATG16L1 (1:50, Abcam, ab195242), anti-cathepsin D (1:50, Santa Cruz Biotechnology, sc377124), anti-p150glued (1:50, BD Biosciences, 610474), anti-STX17 (1:50, Abcam, ab229646) and anti-SMI 32 (1:500, BioLegend, 801701).

Techniques: Western Blot