Journal: Biomolecules

Article Title: Cholesterol Modulation Attenuates the AD-like Phenotype Induced by Herpes Simplex Virus Type 1 Infection

doi: 10.3390/biom14050603

Figure Lengend Snippet: Summary of antibodies employed in Western blot (WB) and immunofluorescence (IF) experiments.

Article Snippet: , Human LAMP2 , Mouse , 1/1000 (WB) 1/50 (IF) , DSHB H4B4.

Techniques: Western Blot, Immunofluorescence, Infection, Plasmid Preparation

Journal: Biomolecules

Article Title: Cholesterol Modulation Attenuates the AD-like Phenotype Induced by Herpes Simplex Virus Type 1 Infection

doi: 10.3390/biom14050603

Figure Lengend Snippet: HSV-1 infection induces intracellular cholesterol accumulation. ( A ) Intracellular cholesterol levels expressed as nanograms of cholesterol per micrograms of protein in SK-N-MC and N2a neuroblastoma cells treated with U18666A and water-soluble cholesterol. ( B ) Intracellular levels of cholesterol in cultures infected with HSV-1 at a multiplicity of infection (MOI) of 10 for 18 h compared to mock-infected cells. The graph data show the mean ± SEM of at least 4 independent experiments. Significance was recorded at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). ( C ) Confocal microscopy images of filipin staining (green) show cholesterol distribution patterns in SK-N-MC and N2a cells infected with HSV-1 (MOI 10) or treated with U18666A. TO-PRO-3-stained nuclei (red) are also shown. Immunofluorescence images of SK-N-MC cells uninfected ( D ) or infected with HSV-1 at MOI 10 ( E ) were obtained with filipin staining (green) and antibodies recognizing different markers (red) of early (EEA1) and late endosomes (CD222), autophagosomes (LC3), and lysosomes (LAMP2). TO-PRO-3-stained nuclei (blue) are also shown. Arrowheads in the merge panels showed the colocalization of filipin with CD222 and LC3 in HSV-1-infected cells. Scale bar: 10 µm.

Article Snippet: , Human LAMP2 , Mouse , 1/1000 (WB) 1/50 (IF) , DSHB H4B4.

Techniques: Infection, Confocal Microscopy, Staining, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Ca 2+ /CaM affects TSC2 localization without altering TSC2 phosphorylation . A , HEK293 cells were starved of serum (−FBS) for 3 h, pretreated with DMSO or BAPTA-AM (50 μM) or W-7 (30 μM) for the last 1 h (BAPTA-AM) or 5 min (W-7) and then stimulated with or without (−) carbachol (100 μM) for 15 min (+CCh). Cell lysates were analyzed by Western blotting with the indicated antibodies. The images of phospho-T389-S6K1 and S6K1 panels in DMSO and BAPTA-AM lanes are reused from Fig. 6 C . B , Quantitation of the relative intensity of phospho-T1462-TSC2 to total TSC2 of (A), in which the carbachol stimulation (DMSO) was set to 1. Graphs represent mean ± SD of three independent experiments. One-way ANOVA with Tukey’s test. C , HEK293 cells were starved of serum (−FBS) for 3 h, and then treated with carbachol (100 μM) or insulin (100 nM) for 15 min. The cells were then immunostained with anti-TSC2 and anti-LAMP2 antibodies. Merged images of TSC2 ( green ), LAMP2 ( magenta ) and nuclei staining with Hoechst 33342 ( blue ) are also shown. Scale bar, 10 μm. D , colocalizations of TSC2 with LAMP2 were quantified, and Pearson’s correlation coefficient is shown. Data from four independent experiments are shown in blue triangles and were used for statistical analysis. Bars represent averages. The values of Pearson’s correlation coefficient of five individual images in each experiment are also shown in gray circles . One-way ANOVA with Tukey’s test, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Phospho-proteomics, Western Blot, Quantitation Assay, Staining