Journal: Journal of Virology

Article Title: CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling

doi: 10.1128/JVI.02251-16

Figure Lengend Snippet: LMP1 trafficking to lipid rafts and downstream signaling do not require CD63. (A) HEK293 control and CD63 knockout cells were transfected with GFP-LMP1. Lipid rafts were biochemically isolated for immunoblot analysis of whole-cell (input) and lipid raft-associated proteins, equal masses loaded. (B) Cytoplasmic and nuclear fractions of HEK293 control and CD63 CRISPR cells transfected with GFP or GFP-LMP1 were separated and confirmed by enrichment of cytoplasmic calnexin or nuclear histone H4 protein, equal masses loaded. (C) Akt and ERK activation in cytoplasmic fractions of HEK293 and CD63 CRISPR cells following GFP-LMP1 transfection was measured, equal masses loaded. Relative levels of phospho-Akt and phospho-ERK were averaged over three independent experiments. (D) HA-LMP1 packaging in EVs (equal volumes) from Rat1 cells stably expressing a pBabe-HA-LMP1 vector following CD63 knockout. (E) Quantitation of HA-LMP1 packaging in Rat1 EVs from three independent experiments. (F) Focus formation assay was performed using Rat1 control or CD63 knockout cells transduced with an empty pBabe vector or pBabe-HA-LMP1. (G and H) Immunoblot analysis of LMP1-induced NF-κB signaling activation in cytoplasmic (G) and nuclear (H) fractions of HEK293 control and CD63 CRISPR cells, equal masses loaded. The results shown are representative of findings from multiple experiments.

Article Snippet: Blots were probed with primary antibodies against the following: Alix (Q-19; Santa Cruz Biotechnology), HSC70 (B-6; Santa Cruz), TSG101 (C-2; Santa Cruz), calnexin (11397; Santa Cruz), caveolin-1 (D46G3; Cell Signaling), flotillin-2 (H-90; Santa Cruz), CD63 (TS63; Abcam), GFP (600-101-215; Rockland), HA (C29F4; Cell Signaling), LMP1 (CS1-4; Dako), histone H4 ( 81 , 82 ), IKKα (11930; Cell Signaling), IKKβ (8943; Cell Signaling), phospho-IKKα/β (2697; Cell Signaling), IκBα (4814; Cell Signaling), phospho-IκBα (2859; Cell Signaling), NF-κB p65 (8242; Cell Signaling), phospho-NF-κB p65 (3033; Cell Signaling), RelB (4922; Cell Signaling), NF-κB p100/p52 (3017; Cell Signaling), phospho-NF-κB p100/p52 (4810; Cell Signaling), ERK 2 (C-14; Santa Cruz), phospho-p44/42 MAPK (9106; Cell Signaling), Akt (9272; Cell Signaling), phospho-Akt (4060; Cell Signaling), and phospho-STAT3 (9134; Cell Signaling).

Techniques: Knock-Out, Transfection, Isolation, Western Blot, CRISPR, Activation Assay, Stable Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Tube Formation Assay, Transduction

Journal: Nucleic Acids Research

Article Title: The GT1 domain of RNase J ensures RNA quality control through dsRNA binding in Arabidopsis plastids

doi: 10.1093/nar/gkag033

Figure Lengend Snippet: RNJ protein domains and structure. ( A ) RNJ linear structures from Arabidopsis thaliana, Populus trichocarpa, Chlamydomonas reinhardtii, Synechocystis sp. PCC 6803, Bacillus subtilis , and Methanocaldococcus jannaschii . Domains drawn to scale are the cTP, chloroplast target peptide; MBL, Metallo-β-lactamase domain; β-CASP, β-CASP domain; CTD, C-terminal domain; GT1, GT1 domain. On the right, protein length is indicated. ( B ) AlphaFold predicted structures as accessible from the AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ). Arabidopsis (At, AF- Q84W56 -F1), Chlamydomonas (Cr, AF-B2YFW5-F1), Synechocystis (Sy, AF- P54123 -F1), and Bacillus (Bs, AF- Q45493 -F1). Domains are colored as in panel (A) and as labeled for AtRNJ. The N-terminal region upstream of the MBL domain is hidden for easier viewing. The proline-rich helix unique to the Chlamydomonas linker region is indicated.

Article Snippet: Spectra were searched against the A. thaliana UniProt reference proteome (Taxon ID 3702, downloaded Jan 5, 2025; 39 396 sequences) supplemented with common contaminants and decoy sequences.

Techniques: Labeling

Journal: Computational and Structural Biotechnology Journal

Article Title: A global view of altered ligand-receptor interactions in bone marrow aging based on single-cell sequencing

doi: 10.1016/j.csbj.2024.06.020

Figure Lengend Snippet: Different LR interactions involving hematopoietic stem cells in old and young bone marrow. A. Left, the number of significantly different LR interactions with hematopoietic stem cells (HSC) as sender cell between the old and young group; Right, the number of significantly different LR interactions with HSC as receiver cell between the old and young group. MS> 0.1 and MS< −0.1 indicate up-regulation and down-regulation in the old group respectively. B．KEGG pathways enriched by receptors in the significantly different LR interactions with HSC as receiver cells. C, D. HEK293T cells were used to overexpress the three ligands Igfbp4, Mmp9 and Selenop respectively. Then the supernatant was then collected to incubate HSCs. WT is the supernatant of wild-type HEK293T without overexpressed protein. Then SPiDER-βGal flow cytometry was used to detect the fluorescence intensity of HSCs (C) and RT-qPCR was used to detect the expression of p16 and p21 in HSCs (D). * P value < 0.05, ** P value< 0.01.

Article Snippet: Mouse Igfbp4 (Refseq ID: 16010), Mmp9 (Refseq ID: 17395), and Selenop (Refseq ID: 20363) were cloned into protein expression plasmids (Sino Biological Inc., Beijing, China).

Techniques: Flow Cytometry, Fluorescence, Quantitative RT-PCR, Expressing